Mutants and disease
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Mutants and Disease. MUPGRET Workshop . Mutation. Heritable change in the DNA sequence. Naturally occurring Induced. Types of mutations. Chromosomal Point Insertion/Deletion DNA repair. Mutagens. Environmental Chemical. Mutations as a tool. Associating a phenotype with a gene.

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Mutants and Disease

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Mutants and Disease

MUPGRET Workshop


Mutation

  • Heritable change in the DNA sequence.

    • Naturally occurring

    • Induced


Types of mutations

  • Chromosomal

  • Point

  • Insertion/Deletion

  • DNA repair


Mutagens

  • Environmental

  • Chemical


Mutations as a tool

  • Associating a phenotype with a gene.

  • Understanding gene function.

  • Studying protein interactions.

  • Understanding cell lineage and organ development.


Associating a phenotype with a gene

  • Changes in the DNA sequence that non-functional or reduced function proteins often cause a visible change in the appearance of the organism.

  • Some changes do not give visible phenotypes.

  • Often identified as an “off-type” in plant species.


Cell may not be able to follow damaged instruction

OR

Damaged protein is made

OR

Spelling error may be harmless

X

X

Damaged protein may or may not be able to function in the cell.

Cell does not make the protein

Functional protein made by the cell

Misspelled Genes: 3 Possible Outcomes

DNA

A misspelled gene


Dwarfing

  • Gibberellic acid (GA) is a plant hormone.

  • GA levels influence growth.

  • Mutants in genes for GA synthesis, reduce plant height.


Associating a phenotype #2

  • This is often the first step towards understanding the function of a gene or to dissecting a biochemical pathway.

  • The mutation can be either a naturally occurring one or an induced one.

  • Can be targeted or random.


Understanding gene function

  • “You don’t know how something really works until you have to fix it.”

  • Disruptions of the gene can be either non-functional or “leaky”.

  • Often the “leaky” phenotypes will really help you understand how to gene works.


Understanding gene function

  • In the case of targeted mutagenesis where you know what the other genes in that would/could be co-regulated with the mutant are you can understand the pathway better by looking at expression of the co-regulated genes.


Understanding gene function

  • In the case of site directed mutagenesis where you can target particular sequences, you can dissect the part of the protein that is important for function.

  • Can help to ID the catalytic site or a site involved in protein-protein interactions or a site involved in transport, etc.


Protein Explorer

  • Protein Explorer http://molvis.sdsc.edu/protexpl/frntdoor.htm

  • Also available at Biology Workbenchhttp://workbench.sdsc.edu/

  • Tutorials at http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/pdb.shtml

  • Troubleshooting http://molvis.sdsc.edu/protexpl/troubles.htm


Studying protein-protein interaction

  • Often use a series of alleles that have defects in different parts of the gene to identify the site that is required for protein-protein interaction.

  • The series can be insertions, deletions, or point mutations and may come from nature or be induced or a combination of the two.


Interaction Maps

  • Molecular Interaction Maps http://discover.nci.nih.gov/mim/index.jsp


Understanding cell lineage

  • Usually used with transposon mutagenesis.

  • Transposons are mobile pieces of DNA that can insert into a gene and disrupt its function.

  • Insertion can happen throughout development and can be used to track where cells came from with visible marker.


Ac/Ds in Maize


Corn example of cell lineage


Methods for detection mutations

  • Alteration in electrophoretic mobility

  • Sequencing

  • Protein trunctation test


Blazing a Genetic Trail

  • It tells the story of how mutations are involved in several different diseases.

  • http://www.hhmi.org/genetictrail/


Association Genetics

  • Usually used for medical genetics.

  • Recently applied to plant genetics.

    • Which genes were involved in domestication?

    • Is this gene responsible for part of the difference we see in a particular trait such as plant height?


Dwarf 8

  • Mutagenesis and trait analysis suggested that d8 might influence flowering time and plant height.


D8 study

  • Sequenced D8 in many ~100 maize lines.

  • Measured flowering time and plant height in the same material.

  • Compare DNA sequence to flowering time and plant height.


D8 summary

  • Found several polymorphisms that are associated with changes in flowering time.

  • Data also indicate that D8 has undergone selection.

    • Compare synonymous vs. nonsynonymous substitutions.


Plants as a Model for Disease

  • Sometimes mutations in the same gene in different organisms have similar phenotype.

  • This allows researchers to choose the organism with the best genetic resources to study the normal function of that gene.

  • This also allows researchers to identify prospective genes for a phenotype in one species, based on another.


Xeroderma pigmentosa

  • Autosomal recessive.

  • UV exposure damages DNA.

  • Defect in DNA damage repair.

  • Risks include cancer, telangiectasia, disfigurement.

  • Can be diagnosed before birth.

  • Take total protection measures from sun/fluorescent light.


Xeroderma pigmentosa


UV damages tissue that contains molecules that can absorb light.


Mechanisms of UV damage

  • Low penetration into tissues.

  • Molecular fragmentation—proteins, enzymes, and nucleic acids contain double bonds that can be ruptured by UV.

  • Free radical generation—molecules of susceptible tissues absorb UV and eject an electron, which is taken up by oxygen, then termed superoxide, a free radical.


Free radicals

  • Are scavenged by superoxide dismutase, vitamin C, vitamin E, glutathione peroxidase, carotene.


Lesion mutant in maize


Prions and Disease

  • Proteins that can change shape.

  • And make other proteins change their shape!

  • As number of changed proteins increases a phenotype is observed.

  • Causal agent of mad cow disease, scrapie in sheep and Creutzfeldt-Jakob disease in humans.


Prions II

  • Previously thought only nucleic acid encoded changes caused disease.

  • Stanley Prusiner discovered prion’s ability to change other protein’s structure and won the Nobel Prize.

  • Sup35 is a prion-like protein in yeast.


Sup35

  • Translation termination factor

  • Carboxyl end binds to the ribosomal complex to terminate translation.

  • If Sup35 is converted to an alternate conformation (infectious prion conformation) the shape change spreads throughout the cell and is passed to daughter cells.


Sup35

  • In prion conformation causes ribosomes to read through stop codons altering shape and function of proteins.

  • Not adaptively advantageous so why is it maintained?


Why?

  • True et al. 2000. Nature 407: 477-483.

  • Reduced translation fidelity, extends proteins.

  • Some of these are antibiotic resistant.

  • Could lead to stabilization of new phenotype under correct environment.


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