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Flashcards in forward genetics Deck (26):
1

the forward genetics approach seeks to..

find the genes encoded by DNA that are responsible for a phenotype of interest

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is forward or reverse genetics the classical genetic strategy

forward

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forward genetics starts with the..

phenotype

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mutants and forwards genetics

mutated genes allow us to figure out which genes responsible for specific phenotypes. e.g. if specific genes are artificially mutated then this will show up as a faulty phenotype and can be identified

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simple process of forward genetics (4)

1. isolate mutants with a phenotype of interest 2. define gene responsible for these phenotypes 3. clone.identify genes 4. analyse the genes to predict encoded proteins

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1.isolate mutants with a phenotype of interest

using model organism

mutagenesis:

  • physical mutagens (UV)
  • chemical (base analogies)
  • biological mutagen

-screen for desired phenotype

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screening for desired phenotype

for essential cell function there are conditional lethal point mutants e.g. temp sensitive mutants -mutant protein is functional at permissive temp -non-function at non-permissive temp

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2.define genes responsible for these phenotypes

gene complementation test an determine if a phentope arises from mutations on the same or separate genes

e.g. white mutants of a normally purple diploid organism -two diff mutant organisms white 1x purple and white 2 x purple both causes a 3:1 purple ration

-however are the mutations present on the same chromosomes e.g. the genes will be on the same chromosome if they do not complement eachother

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3. clone./identify the genes

cloning by mutant rescue e.g. using a gene library to transform cells. Then isolate transformants which rescue or restore a wild-type phenotype e.g. identify cells that have the mutation and clone

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mutant rescue

using a gene library to transform cells. Then isolate transform-ants which rescue or restore a wild-type phenotype

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cell cycle and gene determination

we can look at cell cycle and determine the role of specific genes- since diff genes have diff roles in the cycle e.g. if one turns off the nda3 gene, G1 will not begin

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gene library

A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA.

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4. analyse the genes to predict encoded proteins

-compare predicted sequence with database sequences

BLAST-basic local alignment search tool- gives clues as to function, which can be tested for

e.g. if you look at the proteins encoded for by genes found at specific points of cell cycle gene libraries can then be used to compare known DNA sequence for genes in one organism to compare to genes in another organism

e.g. when they used s.pombe to find the CDC2 mutant, they also discovered it in the human cDNA library

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CDC2

found in all eukaryotes snd produces cyclin dependent kinases, which control cell cycle

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s.pombe experiment and gene library and the discovery of CDC2

s.pombe double stranded DNA is cleaved by restriction endonuclease--> cut into millions of tiny little segments and inserted into plasmids. -these recombinant plasmids are then introduced into bacteria--> genetic library for s.pombe mutant rescue in S.pombe to discover CDC2 gene

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mutant rescue in s.pombe

conditional lets s.pombe (temp sensitive) is crossed with s.pombe wild top library in plasmid replicating vector. next: transform and selection those cells which survive at 36 degrees have their plasmids recovered (in e.coli) and this gives us a cloned CDC2 gene

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what principally control cell cycle

protein phosphorylation--> therefore controlled by CDKs (cyclin dependent kinases)

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cdk

cyclin dependent kinases

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how were CDKs discovered

by genetic analysis of the cell cycle

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what can we use to estimate relatedness of species

phylogenetics

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phylogenetics can be used to

estimate the relatedness between species based on sequence data

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forward genetics has been used to..

understand relationships e.g. using BLAST similarities between organismic can be found and similarity often infers homology e..g. if genes are similar then similar proteins will be coded for

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divergence between two homologues sequences is proportional to

mutation/recombination rate

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phylogenetic can be examined using

phylogeny trees

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BLAST

BASIC LOCAL ALIGNMENT SEARCH TOOL -BLAST finds regions of similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance.

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orfs found in mutant rescue

(ORF) is the part of a reading frame that has the potential to be translated