Download Lect 6 JF 2012.pptx

Lecture 6
The connection between genes,
proteins and metabolism
CAMPBELL BIOLOGY
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What do genes do?
What cellular processes do they affect?
1902 - Garrod described: inherited disorders of metabolism
also called: Inborn Errors of Metabolism
He was studying the disorder called alkaptonuria
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affected individuals produce urine that turns black
the disorder runs in families – inherited as a recessive trait
affected individuals have a metabolic block preventing:
homogentistic acid
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maleyl aceto acetic acid
homogentistic acid accumulates and is excreted in urine
is not a life-threatening condition
Phenylketonuria (PKU) - a more serious inherited defect
-  a defect in the metabolism of the amino acid: phenylalanine
phenylalanine
tyrosine
-  leads to accumulation of a toxic metabolite = phenylpyruvic acid
-  if untreated, can cause severe mental retardation
-  all newborns are routinely tested for PKU and other inherited
metabolic disorders = the Guthrie heel prick test a single drop
of blood is assayed
-  PKU is a recessive genetic disorder
-  frequency of occurrence = 1/ 11,000 births
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The discovery of Inborn Errors of Metabolism indicated that
“genes control metabolism”
How? Beadle and Tatum (1940’s) proposed:
- genes control the production of (or code for) enzymes
that function in metabolic pathways
But what is the nature of the relationship between
genes and enzymes?
Does one gene code for one enzyme?
Does one gene code for several enzymes?
Do several genes code for one enzyme?
Beadle and Tatum proposed:
that ONE GENE codes for ONE ENZYME
Neurospora crassa
This is known as the ONE GENE : ONE ENZYME hypothesis
- they studied “nutritional mutants” = auxotrophs
of the red bread mould Neurospora crassa
- the wild-type fungus can grow on minimal medium
- auxotrophs can’t grow on minimal medium
they require extra nutritional supplements
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• 
The WILD-TYPE fungus can make all the complex
biochemicals necessary for life (amino acids, vitamins,
etc) from Minimal Medium (MM):
(a) inorganic salts: nitrate, phosphate, potassium, etc
(b) Carbon source: sugar
• 
AUXOTROPHS are unable to make certain biochemicals
and therefore require MM + supplements:
(c) amino acids (20)
(d) vitamins
(e) purines
(f) pyrimidines
required for protein synthesis
required as enzyme cofactors
precursors for DNA/RNA
precursors for DNA/RNA
Beadle and Tatum’s experiments:
1. Create mutants by exposing fungal spores to radiation
X-rays
2. Pick up individual spores and grow them in test-tubes containing
complete medium
Result:
Both mutant and wild-type
spores grow well
3. Transfer fungus to minimal medium
Result:
The wild-type grows well
but the mutant can’t
grow
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Why can’t the mutant grow on minimal medium?
- because it can’t make an essential biochemical
Which essential biochemical can’t it make? i.e.
What nutritional supplement when added to minimal
medium will allow it to grow?
Experiment 1
Complete
Medium
MM
MM
MM
MM
+ vitamins + purines + amino acids
NO GROWTH
GROWTH
Conclusion: the mutant can’t make one or more amino acids
Beadle &
Tatum
http://farm5.static.flickr.com/4147/4997178153_2e39c95285.jpg
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Experiment 2
  Which one of 20 amino acids can the mutant not make?
  Inoculate mutant onto MM + each amino acid
MM
+ glycine
MM
+ alanine
MM
+ serine
MM
MM
+ tyrosine + arginine
NO GROWTH
GROWTH
Conclusion: supplying arginine allows the mutant to grow
therefore the mutant can’t make arginine itself
Beadle &
Tatum
http://farm5.static.flickr.com/4107/4997178187_9d1214af9f.jpg
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Can these studies tell us anything about the metabolic
pathway responsible for biosynthesis of arginine ? YES
  Beadle and Tatum went on to identify 3 different classes
of mutants that could not synthesize arginine
  Each mutant class had a metabolic block at a different step
in the metabolic pathway that produces arginine
  Because they isolated 3 classes of mutants that couldn’t
synthesize arginine, this suggested that 3 genes were involved
in arginine biosynthesis
i.e. that the metabolic pathway contained 3 enzymatic steps
 Metabolic pathways usually involve multiple enzymatic steps
 Each unique enzyme is encoded by a unique gene
?
?
?
arginine
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Class C mutants
- can’t grow if supplied with ornithine nor citrulline
- can only grow if they are supplied with arginine
- therefore the metabolic block must be in the last enzymatic
step that converts citrulline
arginine
MM Orn Cit Arg
Class B mutants
-  can’t grow if supplied with the ornithine
-  but can grow if they are supplied with citrulline or arginine
-  therefore the enzymatic block must be in the enzymatic step that
converts ornithine
citrulline
MM Orn Cit Arg
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Class A mutants
- Will grow if supplied with either ornithine or citrulline or arginine
- Therefore the metabolic block must lie upstream of ornithine
MM Orn Cit Arg
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Beadle and Tatum’s experiments showed:
1.  It is possible to work out the order in which the enzymatic
steps occur in a metabolic pathway using a genetic
approach
2.  That one gene codes for one enzyme
3.  This definition was modified when it was discovered that many
genes code for proteins that are not enzymes e.g. hemoglobin
one gene codes for one protein
4.  It was modified again when it was discovered that some
proteins contain more than one polypeptide chain each of
which is encoded by a separate gene e.g. hemoglobin
one gene codes for one polypeptide
Beadle and Tatum won the Nobel Prize in 1958
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geneticsofmd.webnode.com/
dwe00212g01 Duchenne muscular dystrophy.gif
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Quick lesson on Introns & Exons – Example DMD gene: a typical eukaryotic gene
is made up of two types of DNA sequence (a) coding sequences called exons and (b)
non-coding sequences called introns . After transcription, introns are removed from
the mRNA (this is called RNA splicing).
(We’ll be learning about promoters early next week)
Transcription (DNA transcribed into RNA)
Promoter
Exon 1
Intron 1 Exon 2
RNA processing
Intron 2
Exon 3
intron removal
+ addition of a polyA tail
Mature mRNA
AAAAAAA
Diffential splicing of RNA can lead to generation of multiple proteins
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Immunocytochemisty of muscle biopsies from untreated patient (DMD),
healthy control (HC) and the 4 treated patients.
New Eng J Med 357:2679 Dec 2007
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Stamer and Stuber 2007
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