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Wild –type Mutant eyeless _lf_

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Wild –type Mutant eyeless _lf_ Powered By Docstoc
					                                                                                                                   How do we Identity Genes
                                                                                                                   Involved in Development?
                                                                                                      Spontaneous Mutations
          BIOL 430 Lecture 6                                                                          Genes were originally defined by mutation and we suggest a
          January 15th 2009                                                                           function for the gene based on the mutant phenotype. For
                                                                                                      example a mutation called eyeless causes flies to develop without eyes.
 Identification of genes by mutation                                                                  Therefore the function of the eyeless gene is required for the formation of eyes.

          (forward genetics)                                                                          Genetic Screens for Mutations
Nature of Mutations –Muller’s Morphs                                                                      Classical “Forward Genetics” :
                                                                                                                              Mutant phenotype                        to               Gene

                                                                                                                 “Reverse Genetics” : Post genomic Era
                                                                                                                      Gene    to      Mutant Phenotype
                                                                                                                                 eg. RNAi (i=interference)
                                                                                                                                 Site directed Gene Knock out
           BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                  BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                                                                                                      Wild –type                                                       Mutant
                                                                                                                                                                       eyeless (lf)
            Wild-type                                                          Antennapedia
                                                                                (Dominant mutation)
           BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                  BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)
http://www.biozentrum.unibas.ch/Research/Cell_Biology/Gehring/Pictures/pic1/pic1.html




pax-6 (eyeless) mutations in mouse and
humans cause absence of eyes




                                                                                                                                                                                      “eyes”



                   Glaser et al. Nature Genetics 7, 463 - 471 (1994)
                                                                                                       http://www.biozentrum.unibas.ch/Research/Cell_Biology/Gehring/Pictures/pictures.html
           BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)       ectopic expression of eyeless
                                                                                                                 BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                                                                                                                                                                                                        1
                                                                                                                 Not all mutations are created equally!
                                                                                                                  allele: One of the variant forms of a genetic
                                                                                                                  locus differing from other forms in
                                                                                                                  its nucleotide sequence.
                                                                                                                 *Point mutations

                                                                                                                 *Chromosome rearrangements
                                                                                                                 (deletions, duplications, translocations,
                                                                                                                 Inversions)

             http://www.biozentrum.unibas.ch/Research/Cell_Biology/Gehring/Pictures/pictures.html                *Transposable elements (jumping genes)
twin of eyeless     (induced to produce eyes on the legs)                                                                   BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)
        BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                         The Nature of Mutations                                                                Development can be sensitive to gene dose
       Wild type                                                                                                   Phenotypes
                                                  +/+                                                                                                                                                Gene Product
                                                                                                                                                                                                     or Activity
       Recessive mutation                         m/m                              X
                                                                                   X
                                                                                                                Extra Wing Veins

                                                                               X
       Dominant Mutations                         M/+                                                           Wild type

       homozygous                          /      /
                                          m/m or +/+
                                                                                                                Wing Notches
       heterozygous                               +/m
       hemizygous                                 0/m                                                           Embryonic Lethality
                                                                                     extra copies of the gene                              0         1      2           4
                                                                                                                                               Gene copy number or dose
       Duplication                               +/+;Dp
                                                 +/Df                   Deletion in the region                    Therefore you can think of mutations as altering
       Deficiency
                                                                                                                  a gene’s activity.
        BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                                BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                                                               Fig. 1.2                                                                                                             Fig. 1.2 cont.

                                                               Different mutations
                                                               that affect the same
                                                               gene are called
                                                               alleles.
                                                               It refers to the state
                                                                 f
                                                               of a gene.


                                                                                                                                                                                 Note: A true dominant
                                                                                                                                                                                 mutation (+/m) =(m/m)
                                                             Most mutations                                                                                                      therefore semi-dominant
                                                             are recessive
        BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)
                                                                                                                            BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                                                                                                                                                                                                                    2
                                                                                                          Dominant alleles show a phenotype in a
                                                                                                               heterozygous state (mut/+)
                                                                                                           These phenotypes can be cause by:
                                                                                                 1. Haploinsufficient genes:
                                                                                                     If one copy of a gene is not sufficient to produce enough
                                                                                                     activity for normal development then the gene is termed
                                                                                                     haploinsufficient.

                                                                                                              How d we k
                                                                                                              H    do                   is haploinsufficient?
                                                                                                                          know a gene i h l i        ffi i ?
                                                                                                              A Deficiency (Df) of the gene in trans to a wild type copy
                                                                                                              should give similar phenotype.
                                                                                                              (eg.    mut/+ = Df/+) Note: most Df/+ have not phenotype

                                                                                                                                                                  Deletion in the region of gene
                                                                                                                              X
                                                                                                                                                       =
                                                                       Fig. 1.13                                            mutant                                       mutant
  Dominant mutations are rare
          BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                                                                                                  Nature of Mutations: HJ Muller’s Morphs (1932)
                                                                                                  Muller, H. J. (1932). Further studies on the nature and causes of gene
 2. Dominant Gain of Function mutations:                                                          mutations. Proc. 6th Inter. Congr. Genet. 1, 213-255.

 These are dominant mutations in non                                                              • H.J. Muller (1946) was awarded Nobel Prize for his

 haploinsufficient genes                                                                          contributions to radiation genetics.

 (eg. Df/+ does not show the phenotype)                                                           • He designated five classes of mutants:
                                                                                                  "amorph, hypomorph, hypermorph, neomorph and
                                                                                                                  p
                                                                                                  antimorph”--"morph"
                                                                                                         p                                      g
                                                                                                                                          meaning "form" –
                                                                Deletion in the region
                                                                                                         It is remarkable how these classifications were
         X
                                             =                                                          made even before the structure of DNA was known
                                                                                                              (Watson and Crick DNA sturcture 1953)
        mutant                                                      Wild type
                                                                                                  These “morphs” were described based on observation of
                                                                                                 phenotypes—but now since we know more about molecular
                                                                                                 DNA structure and regulation we can give some examples or
                                                                                                          explanations for the mutant phenotypes.
          BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




  Recessive (loss of function) :
Amorph or null: complete loss of gene function, for example a gene deletion or
early stop codon Genetic criteria: mut/mut = mut/Df                                                        Recall
(strongest in an allelic series, not usually conditional i.e. temp. sensitive)
Note it does not imply anything about the molecular lesion
(sequencing of allele will determine if it is a molecular null)
                                                                                                   2. Dominant Gain of Function mutations:
                                                                                                   These are dominant mutations in non
                      X                                     X
                      X                  =                                                         haploinsufficient genes
             mutant phenotype                              mutant phenotype
                                                                                                   (eg. Df/+ does not show the phenotype)
 Phenotype is similar when compared to a Df
Hypomorph: Partial loss of function. Sometimes referred to as “leaky mutation”
For example temperature sensitive mutant, missense mutation that still has function                                                                             Deletion in the region
such as a protein that is less stable than the wild type protein.
                                                                                                          X
Genetic criteria: mut/mut < mut/Df                                                                                                              =
                  X
                  X                    <               X

                                                                                                         mutant                                                        Wild type
             mutant phenotype                     stronger mutant phenotype

     Phenotype is less when compared to a Df
          BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                                                                                                                                                                                                     3
           Dominant (Gain-of-function) mutations                                                            Antimorph or Dominant Negative: A decrease in wild
            hypermorph, antimorph, neomorph:                                                                type gene activity. Always dominant.
                                                                                                            Poison product. For example: a Receptor that normally
  Hypermorph: An increase in wild type gene activity. Examples
  include a protein that is more active, more stable or is made at higher
                                                                                                            forms dimers to be active. A truncated version of the
  levels. Some hypermorhpic mutations may be recessive.                                                     receptor may act as dominant negative because it can still
  Genetic criteria: mut/mut>> mut/+ >> mut/Df or adding duplications or                                     complex with the wild type receptor and this heterodimer
  extra copies = mut/mut                                                                                    may not be functional.

                                                                                                            Genetic criteria: mut/Df>> mut/+>> mut/+++
      X
      X                       >>          X
                                                                  >           X


strongest mutant phenotype                                          weaker mutant phenotype
                                                                                                                  X
                                                                                                                                        >>           X
                                                                                                                                                                             >                X



       or                                                                                                   Strongest phenotype                                                                    +
                         Add extra copies
                         makes phenotype                                                                                                                                             Weakest phenotype
                         worse
                                                                                                          Adding wild type copies alleviates phenotype
            BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                      BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




A mutation that gives rise to a                      Normally the receptor is activated by a
truncated receptor can act as a                      ligand and dimerizes to signal. A
                                                                                                          Neomorph: A new or novel function of the gene.
dominant negative mutation                           truncated receptor can bind with wild                Such as mutations that cause the gene to be
                                                     type receptor but cannot signal
                                                                                                          expressed at different times or different tissues during
                                     X                                                                    development. Ectopic expression. For example change
                                                                                                          in promoter specificity.
                                                                                                                        wild type promoter
                                                                                                                                                                               gene
                                                                                                          For example normally this promoter expresses this gene in the future eye tissue


                                                                                                          mutation changes promoter specificity

                                                                                                                                           x                                    gene
                                                                                                           This promoter mutation now causes other transcription factors to bind and
                                                                                                           activate transcription in tissues that normally doesn’t express this gene.

                                                                                                            Some of the most interesting mutations are neomorphic mutations
                                                                                                            for example Antennapedia
Fig. 3.59                                                                                                             BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                  Complementation Test                                                                                outcome 1                                              outcome 2
How do you know you have identified a different
                                                                                                                          X                                                          X
gene in a common developmental pathway?                                                                                    Z                                                                                 X

Situation: I have two mutant fly strains that have
an identical eyeless phenotype. Do both strains                                                           eyeless phenotype                                         wild-type phenotype
have mutations that affect the same gene?                                                                 (non complementation)                                     (complementation)

   strain 1:                                             strain 2:                                        Therefore the                                           Therefore the
                                                                                                   X
       X
       X
                                              Z
                                              Z
                                                               or                                  X      mutation affects the                                    mutation affects a
    eyeless phenotype               eyeless phenotype                 eyeless phenotype
                                                                      linked mutation in different gene
                                                                                                          same gene                                               different gene that
                                    mutation in same gene
                                                                      (linked = same chromosome)          -note there are some rare                               functions in the
                                                                                                          exceptions that show intergenic
 Cross the strains to make heterozygous for                                                               non complementation                                     same process.
 for both alleles:
            BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)                      BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




                                                                                                                                                                                                                 4
How do identify and isolate
 genes in development?




 BIOL 430 Molecular Genetics of Development 2009 Dr. Ian Chin-Sang (www.chin-sang.ca)




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