Date post: | 19-Dec-2015 |
Category: |
Documents |
View: | 213 times |
Download: | 0 times |
Dissection of gene function: Dissection of gene function: mutational analysismutational analysis
A.A. Using Model organisms Using Model organisms
B.B. Defining the genesDefining the genes
1.1. Forward GeneticsForward Genetics
2.2. Reverse GeneticsReverse Genetics
A. Using Model organismsA. Using Model organisms
Defining a Model – Defining a Model – Organisms suitable for genetic Organisms suitable for genetic experimentationexperimentationhttp://www.exploratorium.edu/imaging_station/gallery.php?Asset=GFP%20%3Cem%3EC.%20elhttp://www.exploratorium.edu/imaging_station/gallery.php?Asset=GFP%20%3Cem%3EC.%20elegans%3C/em%3E&Group=&Category=%3Ci%3EC.%20elegans%3C/i%3E&Section=Introductiegans%3C/em%3E&Group=&Category=%3Ci%3EC.%20elegans%3C/i%3E&Section=Introductionon
Keys to a suitable genetic modelKeys to a suitable genetic model1.1. Short life cycleShort life cycle
2.2. Mating must produce large number of Mating must produce large number of offspringoffspring
3.3. Easy/inexpensive to handleEasy/inexpensive to handle
4.4. Genetic variation must exist among the Genetic variation must exist among the individuals in the populationindividuals in the population
Model organisms:Model organisms:
Saccharomyces cervisiaeSaccharomyces cervisiae - - yeastyeastDrosophila melanogaster – Drosophila melanogaster – fruit flyfruit flyCaenorhabditis elegans – Caenorhabditis elegans – nematode wormnematode wormArabidopsis thaliana – Arabidopsis thaliana – mustard weed mustard weed Mus musculus – Mus musculus – house house mousemouse
Yeast – genes easily manipulated due to their haploid/diploid lifecycle. Can easily detect recessives in haploid cells, and diploid makes complementation tests possible.
DrosophilaDrosophila
P element = 2907-bp sequence features a perfect 31-bp terminal inverted repeat and an 11-bp subterminal inverted repeat for efficient transposition, along with other repeat units of unknown function plus a transposase gene. Can be used to assist in cloning (insertion mutations).
Balancer chromosome – prevents the recovery of crossover products, so together w/ it’s homologous normal chromosome – they act like a haploid, no recombinants passed on to progeny.Progeny carrying chromosomes that are the products of recombination between balancer and normal chromosomes are not viable.
Mouse – most “relevant and accessible” Mouse – most “relevant and accessible” model to humansmodel to humans
Rapid reproduction, easy Rapid reproduction, easy to rear in the labto rear in the lab
Similar body plans, similar Similar body plans, similar developmentdevelopment
Genomes similar in sizeGenomes similar in size Most human genes have Most human genes have
homologs in micehomologs in mice Similar linkage patternsSimilar linkage patterns
Unable to perform large Unable to perform large scale genetic screens scale genetic screens using mice, but useful in using mice, but useful in determining the function & determining the function & regulation of specific regulation of specific genesgenes
Contributions:Model for human diseases
Cancer geneticsImmunogenetics
B. Defining the genesB. Defining the genes
Goal of Goal of genetic dissectiongenetic dissection = discover all = discover all the genes that affect a phenotype and the genes that affect a phenotype and determine how the genes functiondetermine how the genes function
Mutational analysesMutational analyses – using crosses & – using crosses & molecular genetic tools to determine gene molecular genetic tools to determine gene functionfunction
Then mutant isolation is followed by Then mutant isolation is followed by defining gene pathways!defining gene pathways!
Complementation TestComplementation Test
Determine if the mutant alleles are alleles Determine if the mutant alleles are alleles of one gene or of different genesof one gene or of different genes
ComplementationComplementation = = production of wild production of wild type phenotype when two recessive type phenotype when two recessive mutant alleles are brought together in mutant alleles are brought together in the same cellthe same cell
Cross two mutant strains and analyze the F1 generation:\There are two alternate outcomes -
case 1 – all offspring are normal
case 2 – all offspring are mutants
Case 2The two recessive mutations affect the same gene and are alleles of one another
Case 1The two mutations are in separate genes and are not alleles of one another. Each F1 is heterozygous at both loci
If two recessive mutations are alleles of the same gene, then the phenotype of an organism that contains one copy of each mutation is mutant; if they are alleles of different genes, then the phenotype of an organism that contains one copy of each mutation is wildtype.
1. Gene dissection: 1. Gene dissection: forward geneticsforward genetics
Classical method, Classical method, mutant huntingmutant hunting::Starts with the wild type genome, exposes it to random Starts with the wild type genome, exposes it to random mutagens and systematically surveys the organism for mutagens and systematically surveys the organism for mutations that share some common phenotypemutations that share some common phenotypeSaturated = mutagenesis is thorough enough that each Saturated = mutagenesis is thorough enough that each gene is mutagenized at least once in the treated gene is mutagenized at least once in the treated populationpopulationChoosing a mutagen – Choosing a mutagen –
UV radiation, good to use on microbes & produces lots of point UV radiation, good to use on microbes & produces lots of point mutationsmutations
Alkylating agents convenient for Drosophila, readily digestedAlkylating agents convenient for Drosophila, readily digested X rays or gamma radiation effective in producing a range of X rays or gamma radiation effective in producing a range of
mutational typesmutational types Transposable elements – can be inserted into a gene, disrupting Transposable elements – can be inserted into a gene, disrupting
the integrity of the exons or regulationthe integrity of the exons or regulation
Selecting & Screening for mutantsSelecting & Screening for mutants
Genetic screen = protocol designed so Genetic screen = protocol designed so that the mutant phenotype can be that the mutant phenotype can be easily identifiedeasily identified
Can choose a range of phenotypes within a Can choose a range of phenotypes within a broad general class.broad general class.
Involves visual examination of large #s of Involves visual examination of large #s of mutagenized organismsmutagenized organisms
Recessive lethals hard to detectRecessive lethals hard to detectConditional mutation, permissive condition & Conditional mutation, permissive condition & restrictive conditionrestrictive condition
Genetic selection = protocol designed Genetic selection = protocol designed to allow only mutants to surviveto allow only mutants to survive
Effective at obtaining one specific type of Effective at obtaining one specific type of mutationmutation
Reduces time/labor required for screensReduces time/labor required for screens Simplest if mutant phenotype enhances Simplest if mutant phenotype enhances
survival under certain conditions (e.g. survival under certain conditions (e.g. antibiotic resistance)antibiotic resistance)
Gene then mapped using techniques described, molecular markers linked to the gene can be used to isolate clones from an existing libraryDNA sequence of selected clone is search for candidate genes (ORFs)Determine which ORF is the target gene!
Only the desired phenotypes evade the filter.
T maze for identifying mutants unable to orient themselves.
Reverse genetics = start with gene, research function
Can use available gene sequences and disrupt their function to assess the role of the normal gene product in the organism, in vivo!1) cDNAs initially identified based on
differential expression – determine protein sequence
2) comparison to database sequences to infer potential function (BLAST)
3) Can use random or Targeted mutagenesis = produce mutation in the gene of interest Gene knock-out Gene replacements
4) analyze the phenotype
2. Gene dissection: 2. Gene dissection: Reverse geneticsReverse genetics
Creating mutantsCreating mutants
1)1) Site directed mutagenesis – mutations induced Site directed mutagenesis – mutations induced at specific locationsat specific locations
If restriction sites known - Use restriction enzymes to If restriction sites known - Use restriction enzymes to cut out short nucleotide sequences and replace cut out short nucleotide sequences and replace them with synthetic oligonucleotide that contains the them with synthetic oligonucleotide that contains the mutated sequencemutated sequence
OR if restriction sites unknown:OR if restriction sites unknown: Oligonucleotide-directed mutagenesisOligonucleotide-directed mutagenesis – ss – ss
oligonucleotide that differs from target sequence oligonucleotide that differs from target sequence only by a few bases can pair w/target DNA, only by a few bases can pair w/target DNA, oligonucleotide acts as a primer to initiate DNA oligonucleotide acts as a primer to initiate DNA synthesis and produce ds molecule w/ a mismatch in synthesis and produce ds molecule w/ a mismatch in the primer regionthe primer region
Oligonucleotide synthesized & then hybridized to SS M13, complementary region anneals, however the non-complementary region doesn’t. DNA pol extends the 3’ end of the oligo to create a DS M13 DNA molecule, ligase seals the deal. The DS molecule is then transformed into E. coli.
Example:Example:
Yeast genome first eukaryotic genome to be sequenced Yeast genome first eukaryotic genome to be sequenced completelycompletely6,200 ORFs, 6,200 ORFs, YKO projectYKO project underway – yeast knock out to underway – yeast knock out to systematically delete each ORF, producing a loss-of-systematically delete each ORF, producing a loss-of-function mutation & then investigate the consequencesfunction mutation & then investigate the consequencesDNA fragment containing kanDNA fragment containing kanRR selectable marker, selectable marker, molecular screen used to determine which transformants molecular screen used to determine which transformants carry the ORF deletion using a PCR-based strategycarry the ORF deletion using a PCR-based strategy
The correct replacement of the gene with The correct replacement of the gene with KanMXKanMX was verified in was verified in the mutants by the appearance of PCR products of the expected the mutants by the appearance of PCR products of the expected size using primers that span the left and right junctions of the size using primers that span the left and right junctions of the deletion module within the genome. Four ORF-specific deletion module within the genome. Four ORF-specific confirmation primersconfirmation primers (A, B, C, and D primers) were chosen for (A, B, C, and D primers) were chosen for each ORF disruption. each ORF disruption.
Amplifying these sequences and hybridizing them to Amplifying these sequences and hybridizing them to microarrays microarrays
Phenotype No. %
Elongate6257
2.70
Round10498
4.59
Small8775
3.68
Large6976
3.29
Football5359
2.54
Clumpy1014
0.55
Other 25 0.57
Total ORFs with phenotypes 673 Total Homozygotes screened 4401 % of ORFs with phenotypes 15.29
Morphological Screen Results
2) Knockout mice 2) Knockout mice (fig. 21.3)(fig. 21.3)
Transgenic approach – complicated process requiring Transgenic approach – complicated process requiring embryonic stem cells (ES)embryonic stem cells (ES)
1)1) Normal gene cloned in bacteria and then “knocked out”, Normal gene cloned in bacteria and then “knocked out”, or disabledor disabled
2)2) neoneo – inserted (confers antibiotic resistance to G418) into – inserted (confers antibiotic resistance to G418) into the middle of the target gene. This disrupts the gene and the middle of the target gene. This disrupts the gene and provides a selectable marker! provides a selectable marker! tktk – second gene, linked to – second gene, linked to disrupted gene (makes cells sensitive to gancyclovir)disrupted gene (makes cells sensitive to gancyclovir)1)1) Targeting vector – contains Targeting vector – contains neo & tkneo & tk
3)3) the disabled gene is transferred to cultured embryonic the disabled gene is transferred to cultured embryonic mouse cells where it may exchange w/the normal mouse cells where it may exchange w/the normal chromosomal copy through homologuos recombinationchromosomal copy through homologuos recombination
4)4) Cells screened by adding G418 to medium & Cells screened by adding G418 to medium & gancycloviergancyclovier
5)5) Surviving cells injected into early stage mouse embryo, Surviving cells injected into early stage mouse embryo, which is implanted into a psuedopregnant mousewhich is implanted into a psuedopregnant mouse
6)6) Cells in embryo carrying the disabled gene and normal Cells in embryo carrying the disabled gene and normal wild-type cells will develop together, producing a wild-type cells will develop together, producing a ChimeraChimera
3) RNA interference3) RNA interference
RNAiRNAi = short RNAs (21-25 nucleotides long) = short RNAs (21-25 nucleotides long) called small interfering RNAs will hybridize with called small interfering RNAs will hybridize with cellular mRNAs that are complementarycellular mRNAs that are complementaryThe bound RNAis are targeted by a protein The bound RNAis are targeted by a protein complex - RISC (RNA induced silencing complex - RISC (RNA induced silencing complex) to destroy those mRNAs by slicing complex) to destroy those mRNAs by slicing them into small pieces.them into small pieces.This nullifies expression of that geneThis nullifies expression of that genehttp://www.pbs.org/wgbh/nova/sciencenow/3210http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html/02.html This This gene silencinggene silencing method has been applied in method has been applied in several model systems to turn off genes!several model systems to turn off genes!
RNA RNA in situin situ hybridization hybridizationcDNA clone labeled with cDNA clone labeled with fluorescent dye, the probe is fluorescent dye, the probe is hybridized to a thin section of hybridized to a thin section of tissuetissue
Presence of stain defines where Presence of stain defines where the gene is expressed at the the gene is expressed at the mRNA levelmRNA level
Expression pattern of a protein Expression pattern of a protein can be analyzed using can be analyzed using immunofluorescence stainingimmunofluorescence staining
Antibody specifically binds to the Antibody specifically binds to the protein of interestprotein of interest
C1-THF synthase in 9.5 day mouse embryo. Tissues that stain specifically are neural tube, vasculature of heart, limb bud, first brachial arch, cranialfacial region, umbilicus, inner ear.
Epithelial cells undergoing apoptosis in mammary gland. Immunofluorescence staining of caspase 3 activity in shed, apoptotic cells (green). Nuclei of surrounding luminal cells are stained in red.