Transcript One Gene - One Enzyme

BIOL 2416
CH 4: Gene Function
How do biochemical reactions take place in a cell?
• Each reaction in a metabolic pathway must be catalyzed by its
own enzyme
• Enzymes are proteins encoded in the cellular DNA (by a gene)
• If the gene is functional, functional enzyme is made, and food
substrate A can be turned into product B:
gene 1
functional enzyme 1
helps convert
A
B
• If the gene has been mutated, the reaction cannot take place in real
time, no functional enzyme is made, and A cannot be turned into
B (mutant cell dies)
• Adding B will overcome the mutation, adding more A will not.
So there is a relationship:
1 gene = 1 enzyme (polypeptide)
Archibald Garrod, 1902
• Alkaptonuria- urine turns black upon
exposure to air
• Inborn error of metabolism
One Gene - One Enzyme Hypothesis
• Discovered by George Beadle and Edward Tatum
(1942)
• Used Neurospora crassa (orange bread mold)
• = HAPLOID mycelial-form fungus (means mutations
cannot be masked)
• Bombarded wild-type cells with X-rays to induce
random mutations in enzyme-coding genes
– Each metabolic pathway involves its own set of enzymes/genes; idea is to
create one mutant for every step (gene) in the pathway
– Isolate all mutants concerning one specific pathway
– Order the mutants to start to discover the gene and enzymes involved in the
pathway
Mutant analysis: first make sure each mutant we study
has one and only one mutated gene
• We cannot study the effects of more than one mutated
gene at a time
• Ensure heritable single gene mutants by performing
specific mating bread mold experiments
• Mate wild type (A) and mutant (a) together:
–
–
–
–
produces A/a ascus
A/a ascus undergoes meiosis and 1 round of mitosis
produces 4 A and 4 a “ascospores
Ascospores can be germinated into 8 babies
• Examine the babies: if 4 babies are wild-type (A), and
4 are mutant (a), by definition there is a mutation in a
SINGLE gene
Life cycle of
Neurospora
crassa
The linear meiosis of Neurospora
Figure 3-10b
Out of the single-gene mutants,
how do we pick the ones in the
same pathway?
Done by testing different
growing conditions (media):
Wild-Type (non-mutated) Neurospora
is prototrophic
Needs only minimal media with:
• Inorganic salts (including Nitrogen source)
• Organic Carbon source (sugar)
• Biotin (a vitamin)
• Minimal media
– Supports growth of wild-type organisms
– Wild-type organisms can make whatever is not
supplied using raw materials in minimal media
– Requires functional genes to make functional
protein enzymes
• Complete media
– Has everything plus the kitchen sink
– Supports wild-type and auxotrophic mutants
– Supplies anything mutants cannot make due to
faulty genes/enzymes
Auxotrophic Mutant
• Mutants are produced by X-ray exposure
• Already seen how potential mutants are mated to wildtype strain to ensure X-ray damage is heritable and in
only one gene
• And how progeny spores that can be germinated
• Auxotrophic mutants can no longer grow on minimal
medium
• In order to grow, mutant needs
– minimal media PLUS particular nutrient supplement to
overcome the gene mutation
– Or can grow on complete medium (not diagnostic)
Method to isolate
auxotrophic
mutations in N.
crassa
How do we pick the correct auxotrophic mutants?
• Consider the biosynthetic pathway:
A>B>C>D>E
– Bread mold needs E to live
– Wild type bread mold can make E from D from C from
B from A in minimal media
– But only if all genes/enzymes are OK
• If any one step (>) in the pathway leading to E is
blocked (due to a mutation in that enzyme gene), no
E is made and the mutant dies on minimal media
• So for this pathway, test mutants to see if they will
live when given E
– If adding E overcomes the mutation: bingo!
– If not, it must be a mutant in a different pathway
E.g. Methionine (E) Biosynthesis
• Involves 4 protein enzymes in a linear pathway
• Encoded by 4 wild-type DNA genes: met-2+, met3+, met-5+, and met-8+:
(met-5+)
(met-3+)
(met-2+)
(met-8+)
enz 1
enz 2
enz 3
enz 4
A
(substrate)
B
C
(intermediates)
D
E
(end
product
Methionine)
Identifying Auxotrophic Mutants in
the E Pathway
• Germinate all spores on complete medium
(so everything, mutant or wild type will
grow)
• Switch to arrays of minimal media with B,
C, D, or E food supplements
• All mutants DIE on minimal media with
NO food supplements
• E pathway mutants die on minimal, but
live on minimal + E supplement
Ordering E pathway mutants:
• Again, all mutants CAN GROW as soon as
supplement E (methionine) is added
• Because supplement E overrides any defects
in “upstream” enzyme genes:
(met-5
A
X
(met-3)
B
(met-2)
( met-8)
X C X D X E
GROW
anyway
Add E
Ordering E pathway mutants:
• Mutants can grow if they receive a downstream supplement to override an
upstream enzyme defect:
• Met-8 mutation can only be overcome by adding E food supplement; means it
must concern the last enzyme/gene in the E pathway
• Met-2 mutation can be overcome by adding D or E; must concern the next-tolast enzyme/gene in the E pathway
• Met-3 mutation can be overcome by adding C, D, or E
• Met-5 mutation can be overcome by adding B, C, D, or E
(met-5) (met-3) (met-2) (met-8)
A
B
C
D
E
(BCDE) (CDE) (DE)
(E)
grow
Genetically based enzyme deficiencies in
humans - usually recessive
• Phenylketonuria (PKU)
–
–
–
–
Cannot convert excess phenylalanine to needed tyrosine
Convert to harmful phenylpyruvic acid instead
Mental retardation, unless on PKU-restricted diet
Guthrie testing on newborns
• Albinism
– Mutated tyrosinase gene prevents normal pigmentation
– No melanin
• Lesh-nyhan syndrome
–
–
–
–
Only males (sex-linked)
Self-mutilation
Eventually fatal
Accumulate excess purines due to purine-processing
enzyme defect
• Tay-sachs disease
–
–
–
–
hexA defect causes ganglioside accumulation in brain
Progressive neurological degeneration
Fatal by age 3-4
Ashkenazi Jews - registry
• Sickle cell anemia
– Hemoglobin defect
– Causes sickling of red blood cells > get
stuck in capillaries > Oxygen deficiency
– Pleiotropic (wide-ranging) effects
– Milder sickle cell “trait”
• Cystic fibrosis
– Abnormal active transport of ions across cell membranes
– Causes thick mucus > must be dislodged
– Eventually fatal
Practice Problem
• Q4.1 A number of auxotrophic mutant
strains were isolated from wild-type,
haploid yeast. These strains responded to
the addition of certain nutritional
supplements to minimal culture medium
either by growth (+) or no growth (0). The
following table gives the growth patterns
for single-gene mutant strains: