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Dissection of gene function: mutational analysis. Using Model organisms Defining the genes Forward Genetics Reverse Genetics. A. Using Model organisms. Defining a Model – Organisms suitable for genetic experimentation

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dissection of gene function mutational analysis
Dissection of gene function: mutational analysis
  • Using Model organisms
  • Defining the genes
  • Forward Genetics
  • Reverse Genetics
a using model organisms
A. Using Model organisms
  • Defining a Model – Organisms suitable for genetic experimentation
  • http://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=Introduction
  • Keys to a suitable genetic model
    • Short life cycle
    • Mating must produce large number of offspring
    • Easy/inexpensive to handle
    • Genetic variation must exist among the individuals in the population
model organisms
Model organisms:
  • Saccharomyces cervisiae - yeast
  • Drosophila melanogaster – fruit fly
  • Caenorhabditis elegans – nematode worm
  • Arabidopsis thaliana – mustard weed
  • Mus musculus – house mouse

Yeast – genes easily manipulated due to their haploid/diploid lifecycle. Can easily detect recessives in haploid cells, and diploid makes complementation tests possible.


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.

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).

mouse most relevant and accessible model to humans
Mouse – most “relevant and accessible” model to humans
  • Rapid reproduction, easy to rear in the lab
  • Similar body plans, similar development
  • Genomes similar in size
  • Most human genes have homologs in mice
  • Similar linkage patterns
  • Unable to perform large scale genetic screens using mice, but useful in determining the function & regulation of specific genes


Model for human diseases

Cancer genetics


b defining the genes
B. Defining the genes
  • Goal of genetic dissection = discover all the genes that affect a phenotype and determine how the genes function
  • Mutational analyses – using crosses & molecular genetic tools to determine gene function
  • Then mutant isolation is followed by defining gene pathways!

Complementation Test

Determine if the mutant alleles are alleles of one gene or of different genes

  • Complementation = production of wild type phenotype when two recessive mutant alleles are brought together in the 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 1

The two mutations are in separate genes and are not alleles of one another. Each F1 is heterozygous at both loci

Case 2

The two recessive mutations affect the same gene and are alleles of one another


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 forward genetics
1. Gene dissection: forward genetics
  • Classical method, mutant hunting:
  • Starts with the wild type genome, exposes it to random mutagens and systematically surveys the organism for mutations that share some common phenotype
  • Saturated = mutagenesis is thorough enough that each gene is mutagenized at least once in the treated population
  • Choosing a mutagen –
    • UV radiation, good to use on microbes & produces lots of point mutations
    • Alkylating agents convenient for Drosophila, readily digested
    • X rays or gamma radiation effective in producing a range of mutational types
    • Transposable elements – can be inserted into a gene, disrupting the integrity of the exons or regulation
selecting screening for mutants
Selecting & Screening for mutants
  • Genetic screen = protocol designed so that the mutant phenotype can be easily identified
    • Can choose a range of phenotypes within a broad general class.
    • Involves visual examination of large #s of mutagenized organisms
      • Recessive lethals hard to detect
      • Conditional mutation, permissive condition & restrictive condition
  • Genetic selection = protocol designed to allow only mutants to survive
    • Effective at obtaining one specific type of mutation
    • Reduces time/labor required for screens
    • Simplest if mutant phenotype enhances survival under certain conditions (e.g. antibiotic resistance)

Gene then mapped using techniques described, molecular markers linked to the gene can be used to isolate clones from an existing library

  • DNA sequence of selected clone is search for candidate genes (ORFs)
  • Determine which ORF is the target gene!

T maze for identifying mutants unable to orient themselves.

Only the desired phenotypes evade the filter.


2. Gene dissection: Reverse genetics

  • 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!
    • cDNAs initially identified based on differential expression – determine protein sequence
    • comparison to database sequences to infer potential function (BLAST)
    • Can use random or Targeted mutagenesis = produce mutation in the gene of interest
      • Gene knock-out
      • Gene replacements
    • analyze the phenotype
creating mutants
Creating mutants
  • Site directed mutagenesis – mutations induced at specific locations
    • If restriction sites known - Use restriction enzymes to cut out short nucleotide sequences and replace them with synthetic oligonucleotide that contains the mutated sequence
      • OR if restriction sites unknown:
    • Oligonucleotide-directed mutagenesis – ss oligonucleotide that differs from target sequence only by a few bases can pair w/target DNA, oligonucleotide acts as a primer to initiate DNA synthesis and produce ds molecule w/ a mismatch in the 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.

  • Yeast genome first eukaryotic genome to be sequenced completely
  • 6,200 ORFs, YKO project underway – yeast knock out to systematically delete each ORF, producing a loss-of-function mutation & then investigate the consequences
  • DNA fragment containing kanR selectable marker, molecular screen used to determine which transformants carry the ORF deletion using a PCR-based strategy
    • The correct replacement of the gene with KanMX was verified in the mutants by the appearance of PCR products of the expected size using primers that span the left and right junctions of the deletion module within the genome. Four ORF-specific confirmation primers (A, B, C, and D primers) were chosen for each ORF disruption.
  • Amplifying these sequences and hybridizing them to microarrays
2 knockout mice fig 21 3
2) Knockout mice (fig. 21.3)
  • Transgenic approach – complicated process requiring embryonic stem cells (ES)
  • Normal gene cloned in bacteria and then “knocked out”, or disabled
  • neo – inserted (confers antibiotic resistance to G418) into the middle of the target gene. This disrupts the gene and provides a selectable marker! tk – second gene, linked to disrupted gene (makes cells sensitive to gancyclovir)
    • Targeting vector – contains neo & tk
  • the disabled gene is transferred to cultured embryonic mouse cells where it may exchange w/the normal chromosomal copy through homologuos recombination
  • Cells screened by adding G418 to medium & gancyclovier
  • Surviving cells injected into early stage mouse embryo, which is implanted into a psuedopregnant mouse
  • Cells in embryo carrying the disabled gene and normal wild-type cells will develop together, producing a Chimera
3 rna interference
3) RNA interference
  • RNAi = short RNAs (21-25 nucleotides long) called small interfering RNAs will hybridize with cellular mRNAs that are complementary
  • The bound RNAis are targeted by a protein complex - RISC (RNA induced silencing complex) to destroy those mRNAs by slicing them into small pieces.
  • This nullifies expression of that gene
  • http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html
  • This gene silencing method has been applied in several model systems to turn off genes!
rna in situ hybridization
RNA in situ hybridization
  • cDNA clone labeled with fluorescent dye, the probe is hybridized to a thin section of tissue
    • Presence of stain defines where the gene is expressed at the mRNA level
  • Expression pattern of a protein can be analyzed using immunofluorescence staining
    • Antibody specifically binds to the protein 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.