Download Lecture#3 Genes encode Proteins Readings: Problems: Concepts

Lecture 3; 2007
Biology 207; Section B2; Good
Lecture#3 Genes encode Proteins
Readings:
Griffiths et al (2004) 8th Edition: pp 185-192; 199-201 (complementation)
Ch. 9: 274-279 (review)
Problems:
Griffiths et al (2004) 8th Edition: Ch. 6: 1-5, 6a, 6b, 25, 28, 50
Concepts:
How are genes and proteins related?
1. Genes can be defined by their mutability
2. Mutations alter a particular function in an organism. -> one gene - one enzyme
3. Changes in a gene -> changes in a protein -> changes in a phenotype.
4. Mutation in different genes can be identified by complementation tests.
We now know that
Genes specify the structure of proteins
Normal (wild type) Gene -> protein -> phenotype
Mutant Gene -> protein -> abnormal phenotype
How did we arrive at this point?
A. Garrod
- early in this century - human hereditary defects were due to defects in basic body
chemistry (biochemistry) -> now called "inborn errors in metabolism"
Focused attention on the metabolic control by genes inherited - ultimately led to the idea
now called: 1-gene: 1-enzyme hypothesis
(Beadle & Tatum 1941) - Nobel Prize
Biosynthetic pathways - Macro molecules are synthesized in a series of steps with each
step catalyzed by an enzyme. Each enzyme is encoded by a gene.
Substrate
Product/Substrate
Product/Substrate
Product
1 -----------------------> 2 ----------------------------> 3 ---------------------------> 4
Enzyme
A
B
C
b+
c+
Gene
a+
Suppose:
If we inactivate (mutate) one gene - say b - in one strain of organism
- in that strain no B enzyme - cannot carry out 2 -> 3 conversion
Therefore no product 3 or 4 is produced -> if product 4 is needed for growth-> no growth
(providing this is the only pathway)
However, if we add back 4 -> growth occurs
-> add back missing compound (3)
-> then it can convert 3 -> 4 (grow again)
- but if add either 1 or 2 -> no growth
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Lecture 3; 2007
Biology 207; Section B2; Good
Experiments of Beadle & Tatum
Work on Neurospora - a fungus with a haploid genome.
Series of steps in their experiments – Fig 6-3 (8th) 9-1(7th)
1 - irradiated wild-type Neurospora -> produce mutations
2 - crossed with wild-type of opposite mating type -> go through fruiting body stage
3 - collect individual ascospores from fruiting body
Result: each spore is a potentially different mutant
4 - culture each single ascospore in tube with
"complete medium" (contains all the compounds needed to grow)
5 - grow up 100's -> 1000's of different spores
6 - test each culture to see if it could grow on
"minimal medium" (contains only those compounds needed for growth and
reproduction of wildtype strains - usually contains only salts and a carbon source – no
AA or vitamins)
Beadle & Tatum found:
1- Many strains that were unable to grow on the minimal medium (MM)
(although they grew fine on complete medium (CM))
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Lecture 3; 2007
Biology 207; Section B2; Good
-> called auxotrophs - a strain that can't grow on MM but can on CM
2- They did test to examine inheritance pattern
- inherited as 1:1 ratio when crossed to wildtype (haploid) 1 mutant: 1 wildtype
- they concluded each was a simple mutation in a single gene (see meiosis section later)
3- They did a test with amino acids or vitamins
- test each amino acid individually
- collect many Arg- auxotrophic mutations
- each an independently produced mutation.
Identified many arg- mutants
-> strains that needed arginine added to the Minimal Medium in order to grow.
Arg mutants provided the focus for Beadle & Tatum's further work
Beadle & Tatum found the many arg mutants mapped to 3 different locations on
separate chromosomes -> therefore there were 3 different genes
Called arg-1, -2, -3
Beadle & Tatum - Also found that the 3 auxotrophs differed in their response to two
chemicals related to arginine; ornithine and citrulline
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Lecture 3; 2007
Biology 207; Section B2; Good
B & T - Knew that cellular enzymes often interconverted related compounds
- proposed biochemical model for Neurospora
precursor--------------> ornithine --------------> citrulline ------------>arginine
Enzyme
A
B
C
arg-2+
arg-3+
Gene
arg-1+
Assume:
1. that a mutation in a particular gene blocks the production of a single enzyme
2. defective enzyme creates a block in the biosynthetic pathway
3. the block can be circumvented by adding the compound that comes after the block
Note: the entire model was inferred from the properties of the mutants (phenotype)
- later the presence of defective enzymes was demonstrated by independent
biochemical analysis
History
- first insight into the function of genes and how they worked
(remember it wasn't until 1944 that DNA was shown to be the genetic material)
Complementation in Diploids
Complementation studies in diploids can be used to demonstrate two mutations are in
either:
− the same gene, or
− two different genes
If two mutations are in different genes then each different gene is associated with a
different enzyme in a biochemical pathway
Previously used Neurospora - haploid organism - only has one copy of each gene
Yeast - can be either haploid or diploid organism
Haploid - simple organism
- can also exist as haploid cells. (like Neurospora)
Assume the following pathway:
Substrate
Product/
Product
Substrate
1 -------------------------> 2 ---------------------------> 3
Enzyme
A
B
Gene
a+
b+
Then in haploid Yeast: a strain can be mutant or wild type at each gene. e.g.:
a+ b+ => wildtype enzymes => growth
a+ b- => mutant enzyme B => no growth
a- b+ => mutant enzyme A => no growth
a- b- => mutant enzymes A &B => no growth
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Lecture 3; 2007
Biology 207; Section B2; Good
Yeast can also exist as a diploid - complementation testing
- two haploid cells come together - fuse two strains
Fuse one haploid strain with another haploid strain
e.g.: First Strain / Second Strain make a Diploid strain
a+ b+
a+ b+
a+ b+ / a+ b+
Having two copies in each cell may permit complementation to take place.
First strain
Second strain
a+ b- and a+ b-
Combined
genotype
a+ b-/a+ b -
Outcome
(phenotype)
No growth
Conclusion:
a-b+ and a- b+
a-b+/a- b+
No growth
No complementation
a- b+ and a+b--
a- b+/ a+b -
Growth
Complementation
No complementation
Complementation:
If the mutations fail to complement then they are mutant in the same gene; they are
allelic mutations.
If the mutations do complement then they are mutant in different genes; they are nonallelic mutations.
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Can be done in diploids using standard crossing techniques
Can also be done in haploids, using either an extra plasmid or creating a diploid
Is commonly done in animals and plants by introducing a new gene through
genetic transformation
Final proof of gene/enzyme function is in almost all cases a complementation test.
See page on Complementation Help in Biol 207 Home page.
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