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RNA
DNA:
double-stranded
deoxyribose
A, C, G, T
RNA:
single-stranded
ribose
A, C, G, U
Complication #5: Can’t add to 5′ end
5′
3′
5′
?
5′
?
3′
5′
Complication #5: Can’t add to 5′ end
how do we replicate this?
5´
5´
3´
5´
5´
new DNA
3´
RNA primer
Complication #5: Can’t add to 5′ end

Multiple primers

Lagging strand made as series of Okazaki fragments
lagging (discontinuous) strand
5´
Okazaki
fragments
5´
3´
5´
5´
5´
leading (continuous) strand
3´
DNA polymerase
Complication #5: Can’t add to 5′ end
3′
3′
3′
5′
origin
5′
3′
Complication #6: DNA contains RNA

DNA polymerase III makes DNA but can’t remove primer
RNA primer
5´
3´
3´
5´
Complication #6: DNA contains RNA

5´
3´
DNA polymerase I replaces RNA with DNA
3´
RNA primer
5´
DNA polymerase I
Complication #6: DNA contains RNA

5´
3´
DNA polymerase I replaces RNA with DNA
5´
Complication #6: DNA contains RNA

5´
3´
DNA polymerase I replaces RNA with DNA
5´
Complication #7: Gaps in the DNA backbone

DNA ligase makes phosphodiester bond
Complication #7: Gaps in the DNA backbone

DNA ligase makes phosphodiester bond
DNA ligase
DNA ligase
What does DNA do?
nucleus
chromosome
Cl–
DNA
molecule
membrane
CFTR
ATP
Cl–
CFTR gene
folding
mRNA
transcription
CFTR
protein
ribosome
The “Central Dogma”
translation
How does a gene encode a protein?
DNA
the “Central Dogma”
of molecular biology
transcription
mRNA
translation
Protein
DNA
Un beau jour, je suis allé au
marché pour acheter du
pain. Il faisait chaud. Alors,
j’ai acheté aussi un
limonade.
transcription
mRNA
Il faisait chaud.
CTACGAGGAGGTGAAGCGATGCCCCGTAGCC
GATAGTAGC
GATGCTCCTCCACTTCGCTACGGGGCATCGGC
gene
TATCATCG
DNA
transcription
5’
3’
mRNA
CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAU
AGUAGC
mRNA
Il faisait chaud.
translation
Protein
It was hot.
CTACGAGGAGGTGAAGCGATGCCCCGTAGCC
GATAGTAGC
GATGCTCCTCCACTTCGCTACGGGGCATCGGC
gene
TATCATCG
DNA
transcription
3’ mRNA
5’ CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAU
AGUAGC
translation
N-MetProArgSerArg-C
protein
The CFTR cDNA is 6,132 nt long. The sequence of one of Sue’s alleles is shown.
 Is Sue a carrier?
AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGAGAGACCATGCAG
AG GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACCAATTTTGAGGAAA
GGATACAGACAGCGCCTGGAATTGTCAGACATATACCAAATCCCTTCTGTTGATTCTGCTGACAATCTAT CTGAAAAATTGGAAAGAGAATGGGATAGAGAGCTGGCTTCAAAGAAAAATCCTAAACTCATTAATGCCCT
TCGGCGATGTTTTTTCTGGAGATTTATGTTCTATGGAATCTTTTTATATTTAGGGGAAGTCACCAAAGCA GTACAGCCTCTCTTACTGGGAAGAATCATAGCTTCCTATGACCCGGATAACAAGGAGGAACGCTCTATCG
CGATTTATCTAGGCATAGGCTTATGCCTTCTCTTTATTGTGAGGACACTGCTCCTACACCCAGCCATTTT TGGCCTTCATCACATTGGAATGCAGATGAGAATAGCTATGTTTAGTTTGATTTATAAGAAGACTTTAAAG
CTGTCAAGCCGTGTTCTAGATAAAATAAGTATTGGACAACTTGTTAGTCTCCTTTCCAACAACCTGAACA AATTTGATGAAGGACTTGCATTGGCACATTTCGTGTGGATCGCTCCTTTGCAAGTGGCACTCCTCATGGG
GCTAATCTGGGAGTTGTTACAGGCGTCTGCCTTCTGTGGACTTGGTTTCCTGATAGTCCTTGCCCTTTTT CAGGCTGGGCTAGGGAGAATGATGATGAAGTACAGAGATCAGAGAGCTGGGAAGATCAGTGAAAGACTTG
TGATTACCTCAGAAATGATTGAAAATATCCAATCTGTTAAGGCATACTGCTGGGAAGAAGCAATGGAAAA AATGATTGAAAACTTAAGACAAACAGAACTGAAACTGACTCGGAAGGCAGCCTATGTGAGATACTTCAAT
AGCTCAGCCTTCTTCTTCTCAGGGTTCTTTGTGGTGTTTTTATCTGTGCTTCCCTATGCACTAATCAAAG GAATCATCCTCCGGAAAATATTCACCACCATCTCATTCTGCATTGTTCTGCGCATGGCGGTCACTCGGCA
ATTTCCCTGGGCTGTACAAACATGGTATGACTCTCTTGGAGCAATAAACAAAATACAGGATTTCTTACAA AAGCAAGAATATAAGACATTGGAATATAACTTAACGACTACAGAAGTAGTGATGGAGAATGTAACAGCCT
TCTGGGAGGAGGGATTTGGGGAATTATTTGAGAAAGCAAAACAAAACAATAACAATAGAAAAACTTCTAA TGGTGATGACAGCCTCTTCTTCAGTAATTTCTCACTTCTTGGTACTCCTGTCCTGAAAGATATTAATTTC
AAGATAGAAAGAGGACAGTTGTTGGCGGTTGCTGGATCCACTGGAGCAGGCAAGACTTCACTTCTAATGG TGATTATGGGAGAACTGGAGCCTTCAGAGGGTAAAATTAAGCACAGTGGAAGAATTTCATTCTGTTCTCA
GTTTTCCTGGATTATGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGA TACAGAAGCGTCATCAAAGCATGCCAACTAGAAGAGGACATCTCCAAGTTTGCAGAGAAAGACAATATAG
TTCTTGGAGAAGGTGGAATCACACTGAGTGGAGGTCAACGAGCAAGAATTTCTTTAGCAAGAGCAGTATA CAAAGATGCTGATTTGTATTTATTAGACTCTCCTTTTGGATACCTAGATGTTTTAACAGAAAAAGAAATA
TTTGAAAGCTGTGTCTGTAAACTGATGGCTAACAAAACTAGGATTTTGGTCACTTCTAAAATGGAACATT TAAAGAAAGCTGACAAAATATTAATTTTGCATGAAGGTAGCAGCTATTTTTATGGGACATTTTCAGAACT
CCAAAATCTACAGCCAGACTTTAGCTCAAAACTCATGGGATGTGATTCTTTCGACCAATTTAGTGCAGAA AGAAGAAATTCAATCCTAACTGAGACCTTACACCGTTTCTCATTAGAAGGAGATGCTCCTGTCTCCTGGA
CAGAAACAAAAAAACAATCTTTTAAACAGACTGGAGAGTTTGGGGAAAAAAGGAAGAATTCTATTCTCAATCCAATCAACTCTATACGAAAATTTTCCATTGTGCAAAAGACTCCCTTACAAATGAATGGCATCGAAGAG
GATTCTGATGAGCCTTTAGAGAGAAGGCTGTCCTTAGTACCAGATTCTGAGCAGGGAGAGGCGATACTGCCTCGCATCAGCGTGATCAGCACTGGCCCCACGCTTCAGGCACGAAGGAGGCAGTCTGTCCTGAACCTGA
TGACACACTCAGTTAACCAAGGTCAGAACATTCACCGAAAGACAACAGCATCCACACGAAAAGTGTCACTG GCCCCTCAGGCAAACTTGACTGAACTGGATATATATTCAAGAAGGTTATCTCAAGAAACTGGCTTGGAAA
TAAGTGAAGAAATTAACGAAGAAGACTTAAAGGAGTGCTTTTTTGATGATATGGAGAGCATACCAGCAGT GACTACATGGAACACATACCTTCGATATATTACTGTCCACAAGAGCTTAATTTTTGTGCTAATTTGGTGC
TTAGTAATTTTTCTGGCAGAGGTGGCTGCTTCTTTGGTTGTGCTGTGGCTCCTTGGAAACACTCCTCTTC AAGACAAAGGGAATAGTACTCATAGTAGAAATAACAGCTATGCAGTGATTATCACCAGCACCAGTTCGTA
TTATGTGTTTTACATTTACGTGGGAGTAGCCGACACTTTGCTTGCTATGGGATTCTTCAGAGGTCTACCA CTGGTGCATACTCTAATCACAGTGTCGAAAATTTTACACCACAAAATGTTACATTCTGTTCTTCAAGCAC
CTATGTCAACCCTCAACACGTTGAAAGCAGGTGGGATTCTTAATAGATTCTCCAAAGATATAGCAATTTT GGATGACCTTCTGCCTCTTACCATATTTGACTTCATCCAGTTGTTATTAATTGTGATTGGAGCTATAGCA
GTTGTCGCAGTTTTACAACCCTACATCTTTGTTGCAACAGTGCCAGTGATAGTGGCTTTTATTATGTTGA GAGCATATTTCCTCCAAACCTCACAGCAACTCAAACAACTGGAATCTGAAGGCAGGAGTCCAATTTTCAC
TCATCTTGTTACAAGCTTAAAAGGACTATGGACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACT CTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCC
AAATGAGAATAGAAATGATTTTTGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTTTAACAACAGG AGAAGGAGAAGGAAGAGTTGGTATTATCCTGACTTTAGCCATGAATATCATGAGTACATTGCAGTGGGCT
GTAAACTCCAGCATAGATGTGGATAGCTTGATGCGATCTGTGAGCCGAGTCTTTAAGTTCATTGACATGC CAACAGAAGGTAAACCTACCAAGTCAACCAAACCATACAAGAATGGCCAACTCTCGAAAGTTATGATTAT
TGAGAATTCACACGTGAAGAAAGATGACATCTGGCCCTCAGGGGGCCAAATGACTGTCAAAGATCTCACA GCAAAATACACAGAAGGTGGAAATGCCATATTAGAGAACATTTCCTTCTCAATAAGTCCTGGCCAGAGGG
TGGGCCTCTTGGGAAGAACTGGATCAGGGAAGAGTACTTTGTTATCAGCTTTTTTGAGACTACTGAACAC TGAAGGAGAAATCCAGATCGATGGTGTGTCTTGGGATTCAATAACTTTGCAACAGTGGAGGAAAGCCTTT
GGAGTGATACCACAGAAAGTATTTATTTTTTCTGGAACATTTAGGAAAAACTTGGATCCCTATGAACAGT GGAGTGATCAAGAAATATGGAAAGTTGCAGATGAGGTTGGGCTCAGATCTGTGATAGAACAGTTTCCTGG
GAAGCTTGACTTTGTCCTTGTGGATGGGGGCTGTGTCCTAAGCCATGGCCACAAGCAGTTGATGTGCTTG GCTAGATCTGTTCTCAGTAAGGCGAAGATCTTGCTGCTTGATGAACCCAGTGCTCATTTGGATCCAGTAA
CATACCAAATAATTAGAAGAACTCTAAAACAAGCATTTGCTGATTGCACAGTAATTCTCTGTGAACACAG GATAGAAGCAATGCTGGAATGCCAACAATTTTTGGTCATAGAAGAGAACAAAGTGCGGCAGTACGATTCC
ATCCAGAAACTGCTGAACGAGAGGAGCCTCTTCCGGCAAGCCATCAGCCCCTCCGACAGGGTGAAGCTCT
TTCCCCACCGGAACTCAAGCAAGTGCAAGTCTAAGCCCCAGATTGCTGCTCTGAAAGAGGAGACAGAAGA
AGAGGTGCAAGATACAAGGCTTTAGAGAGCAGCATAAATGTTGACATGGGACATTTGCTCATGGAATTGG
AGCTCGTGGGACAGTCACCTCATGGAATTGGAGCTCGTGGAACAGTTACCTCTGCCTCAGAAAACAAGGA TGAATTAAGTTTTTTTTTAAAAAAGAAACATTTGGTAAGGGGAATTGAGGACACTGATATGGGTCTTGAT
AAATGGCTTCCTGGCAATAGTCAAATTGTGTGAAAGGTACTTCAAATCCTTGAAGATTTACCACTTGTGT TTTGCAAGCCAGATTTTCCTGAAAACCCTTGCCATGTGCTAGTAATTGGAAAGGCAGCTCTAAATGTCAA
TCAGCCTAGTTGATCAGCTTATTGTCTAGTGAAACTCGTTAATTTGTAGTGTTGGAGAAGAACTGAAATC ATACTTCTTAGGGTTATGATTAAGTAATGATAACTGGAAACTTCAGCGGTTTATATAAGCTTGTATTCCT
TTTTCTCTCCTCTCCCCATGATGTTTAGAAACACAACTATATTGTTTGCTAAGCATTCCAACTATCTCAT TTCCAAGCAAGTATTAGAATACCACAGGAACCACAAGACTGCACATCAAAATATGCCCCATTCAACATCT
AGTGAGCAGTCAGGAAAGAGAACTTCCAGATCCTGGAAATCAGGGTTAGTATTGTCCAGGTCTACCAAAA ATCTCAATATTTCAGATAATCACAATACATCCCTTACCTGGGAAAGGGCTGTTATAATCTTTCACAGGGG
ACAGGATGGTTCCCTTGATGAAGAAGTTGATATGCCTTTTCCCAACTCCAGAAAGTGACAAGCTCACAGA CCTTTGAACTAGAGTTTAGCTGGAAAAGTATGTTAGTGCAAATTGTCACAGGACAGCCCTTCTTTCCACA
GAAGCTCCAGGTAGAGGGTGTGTAAGTAGATAGGCCATGGGCACTGTGGGTAGACACACATGAAGTCCAA GCATTTAGATGTATAGGTTGATGGTGGTATGTTTTCAGGCTAGATGTATGTACTTCATGCTGTCTACACT
AAGAGAGAATGAGAGACACACTGAAGAAGCACCAATCATGAATTAGTTTTATATGCTTCTGTTTTATAAT TTTGTGAAGCAAAATTTTTTCTCTAGGAAATATTTATTTTAATAATGTTTCAAACATATATAACAATGCT
GTATTTTAAAAGAATGATTATGAATTACATTTGTATAAAATAATTTTTATATTTGAAATATTGACTTTTT ATGGCACTAGTATTTCTATGAAATATTATGTTAAAACTGGGACAGGGGAGAACCTAGGGTGATATTAACC
AGGGGCCATGAATCACCTTTTGGTCTGGAGGGAAGCCTTGGGGCTGATGCAGTTGTTGCCCACAGCTGTA TGATTCCCAGCCAGCACAGCCTCTTAGATGCAGTTCTGAAGAAGATGGTACCACCAGTCTGACTGTTTCC
ATCAAGGGTACACTGCCTTCTCAACTCCAAACTGACTCTTAAGAAGACTGCATTATATTTATTACTGTAA GAAAATATCACTTGTCAATAAAATCCATACATTTGTGTGAAA
How to read DNA: the Genetic Code
the Genetic Code

Shown as mRNA

5′ → 3′

64 codons

Redundant

One “start” codon: AUG

Three “stop” codons: UAG,
UAA, UGA (don’t encode
amino acids)
AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGC
ACCCAGAGTAGTAGG
TCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGA
GAGACCATGCAGAG
GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACC
AATTTTGAGGAAA
AATTGGAAGCAAATGACATCACAGCAGGTCAGAGAAAAAGGGTTGAGCGGCAGGC
ACCCAGAGTAGTAGG
AspTrpLys…
IleGlySer…
LeuGluAla…
TCTTTGGCATTAGGAGCTTGAGCCCAGACGGCCCTAGCAGGGACCCCAGCGCCCGA
GAGACCATGCAGAG
GTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCTGGACCAGACC
AATTTTGAGGAAA
the Genetic Code

For each gene, one DNA strand is transcribed
 Template strand “read”
 mRNA complementary to template
5’ CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC 3’
DNA 3’ GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG 5’
template
strand
5’ CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’
mRNA
the Genetic Code

Non-template strand looks like RNA (except T’s)
5’ CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGC 3’
DNA 3’ GATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG 5’
5’ CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’
non-template
strand
mRNA
the Genetic Code

Ribosome starts at 5’ end of mRNA (in eukaryotes)

Protein synthesis begins at start codon, ends at stop codon
codons
5’ CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’
MetProArgSerArg
startthis
codon:
stop codon stop codon:
AUG
UAG, UGA or UAA
is
not “in-frame”
so it’s ignored!
mRNA
protein
What about
Mutation

Change in nucleotide sequence of DNA

Caused by:
 DNA polymerase mistake
 Chemical change within cell
 UV light or other radiation
 Mutagenic chemical
Effects of mutations
DNA
5’
ACGATCCTATGCCCCGCAGCCGATAGTCTCATT 3’
3’ TGCTAGGATACGGGGCGTCGGCTATCAGAGTAA 5’
5’
RNA
ACGAUCCUAUGCCCCGCAGCCGAUAGUCUCAUU
MetProArgSerArg
3’
Effects of mutations
DNA
5’
ACGATCCTATGCCCCGCCGCCGATAGTCTCATT 3’
3’ TGCTAGGATACGGGGCGGCGGCTATCAGAGTAA 5’
5’
RNA
ACGAUCCUAUGCCCCGCAGCCGAUAGUCUCAUU
MetProArgSerArg
3’
Effects of mutations
DNA
5’
ACGATCCTATGCCCCGCCGCCGATAGTCTCATT 3’
3’ TGCTAGGATACGGGGCGGCGGCTATCAGAGTAA 5’
5’
RNA
ACGAUCCUAUGCCCCGCCGCCGAUAGUCUCAUU
MetProArgSerArg
3’
Effects of mutations
Missense: single amino-acid change
DNA
5’
ACGATCCTATGCCCCGCCGCCGATAGTCTCATT 3’
3’ TGCTAGGATACGGGGCGGCGGCTATCAGAGTAA 5’
5’
RNA
ACGAUCCUAUGCCCCGCCGCCGAUAGUCUCAUU
MetProArgArgArg
Could this change have an effect on protein function?
Could it affect the whole organism?
3’
Effects of mutations
Missense: single amino-acid change
Wild-type RPE65:
ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAAACTGTTTGAAACTGTGGAGGAACTGTCCT
CGCCGCTCACAGCTCATGTAACAGGCAGGATCCCCCTCTGGCTCACCGGCAGTCTCCTTCGATGTGGGCC
AGGACTCTTTGAAGTTGGATCTGAGCCATTTTACCACCTGTTTGATGGGCAAGCCCTCCTGCACAAGTT…
MetSerIleGlnValGluHisProAlaGlyGlyTyrLysLysLeuPheGluThrValGluGluLeuSerSerPro
LeuThrAlaHisValThrGlyArgIleProLeuTrpLeuThrGlySerLeuLeuArgCysGlyProGlyLeuPhe
GluValGlySerGluProPheTyrHisLeuPheAspGlyGlnAlaLeuLeuHisLysPheAspPheLysGlu…
Mutant RPE65:
ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAAACTGTTTGAAACTGTGGAGGAACTGTCCT
CGCCGCTCACAGCTCATGTAACAGGCAGGATCCCCCTCTGGCTCACCAGCAGTCTCCTTCGATGTGGGCC
AGGACTCTTTGAAGTTGGATCTGAGCCATTTTACCACCTGTTTGATGGGCAAGCCCTCCTGCACAAGTT…
MetSerIleGlnValGluHisProAlaGlyGlyTyrLysLysLeuPheGluThrValGluGluLeuSerSerPro
LeuThrAlaHisValThrGlyArgIleProLeuTrpLeuThrSerSerLeuLeuArgCysGlyProGlyLeuPhe
GluValGlySerGluProPheTyrHisLeuPheAspGlyGlnAlaLeuLeuHisLysPheAspPheLysGlu…
Effects of mutations
Missense: single amino-acid change
Hemoglobin → sickle-cell anemia:
ATGGTGCATCTGACTCCTGAGGAG…
MetValHisLeuThrProGluGlu
GTG
Val
Effects of mutations
AUGCCCCGCAGCCGAUAG
MetProArgSerArg
RNA
protein
Effects of mutations
Nonsense: creates stop codon
AUGCCCCGCAGCCGAUAG
MetProArgSerArg
AUGCCCCGCAGCUGAUAG
MetProArgSerXXX
Effects of mutations
Deletion: creates frameshift, changes all following aa’s
AUGCCCCGCAGCCGAUAG
MetProArgSerArg
AUGCCCGCAGCUGAUAG
MetProAlaAlaAsp…
Effects of mutations
Insertion: creates frameshift, changes all following aa’s
AUGCCCCGCAGCCGAUAG
MetProArgSerArg
AUGGCCCCGCAGCUGAUAG
MetAlaProGlnProIle…
Effects of mutations
Silent: DNA changes, but protein doesn’t
AUGCCCCGCAGCCGAUAG
MetProArgSerArg
AUGCCCCGCAGUCGAUAG
MetProArgSerArg
What is a gene?

Responsible for an inherited character

Physical unit passed from parent to child in the gamete

Can occur in various “forms” (alleles)

A segment of a DNA molecule = segment of chromosome

Encodes a protein (polypeptide)
Garrod: genes specify enzymes

“Inborn errors of metabolism”

Alkaptonuria
 Urine turns black (“blackwater”)
 Homogentisic acid (“alkaptan”)
Archibald Garrod (1902)
Garrod: genes specify enzymes

Molecules are made by metabolic pathways
 Enzymes catalyze each step
 Missing enzyme → accumulation of preceding molecule
E
E
A
Enzyme 1
B
Enzyme 2
C
Enzyme 3
D
E
Enzyme 4
E
E
Enzyme 5
E
F
Garrod: genes specify enzymes

Hypothesis: alkaptonuria results from missing enzyme
 Pathway blocked, homogentisic acid accumulates
excreted in urine
Beadle and Tatum: One gene, one enzyme

“One ought to be able to discover what genes do by making
them defective”
George Beadle and Edward Tatum (1941)
Neurospora crassa (bread mold)
Beadle and Tatum: One gene, one enzyme

Neurospora grows on minimal medium
 Makes all needed amino acids, nuclotides, etc. from glucose
minimal
medium
(glucose & salts)
Beadle and Tatum: One gene, one enzyme

X-rays produce mutations (changes in DNA)

Some mutants are auxotrophs
 can’t make all needed molecules
 can’t grow on minimal medium
wildtype
auxotrophic
mutant
minimal
medium
Beadle and Tatum: One gene, one enzyme

What’s wrong with the mutant?
minimal
medium
auxotrophic mutant grown on
minimal medium plus:
met
leu
ala
arg
val
phe
Beadle and Tatum: One gene, one enzyme

Mutation “blocks” metabolic pathway at one step
ornithine
citrulline
arginine
mutant #1
minimal
medium
minimal
medium
+ornithine
minimal
medium
+ citrulline
minimal
medium
+ arginine
Beadle and Tatum: One gene, one enzyme

Mutation “blocks” metabolic pathway at one step
ornithine
citrulline
arginine
mutant #2
minimal
medium
minimal
medium
+ornithine
minimal
medium
+ citrulline
minimal
medium
+ arginine
Beadle and Tatum: One gene, one enzyme

What is it that’s “blocked” in the mutants?
 Lack an enzyme in the pathway
 Enzyme is missing because gene is defective
ornithine
enzyme
#1
gene 1
citrulline
enzyme
#2
gene 2
arginine
Beadle and Tatum: One gene, one enzyme
Hypothesis:
 One gene is responsible for making one enzyme
 One enzyme carries out one metabolic step
ornithine
enzyme
#1
gene 1
citrulline
enzyme
#2
gene 2
arginine
Mutation

Change in nucleotide sequence of DNA

Caused by:
 DNA polymerase mistake
 Chemical change within cell
 UV light or other radiation
 Mutagenic chemical
Mutations create new alleles!

Changes in DNA are inherited

Neutral, harmful or beneficial

“Raw material” of evolution
round seed
allele
wrinkled seed
allele
normal
CFTR
allele
brown hair
pigment
allele
non-functional
CFTR
allele
(CF)
blonde
(no pigment)
allele
Why does
SMOKING
cause cancer?
Cancer starts with uncontrolled cell division

Mutation removes normal
growth control

Further mutations allow
cancer cells to spread and
invade
Bone marrow from
a leukemia patient
Smoking increases the mutation rate

Cigarette smoke contains mutagens

Increased frequency of all mutations

Some can affect cell division
Carcinogens = cancer-causing chemicals

Nearly always mutagens













Formaldehyde
Hydrazine
Vinyl chloride
Urethane
2-Nitropropane
Quinoline
Benzo[a]pyrene
Dibenz[a,h]anthracene
Benzo[b]fluoranthene
Indeno[1,2,3-cd]pyrene
Chrysene
Methylfluoranthene
Nitrosodimethylamine












Nitrosoethymethylamine
Nitrosodi-n-butylamine
Nitrosopiperidine
N'-Nitrosoanabasine
Dibenzo[c,g]carbazole
Polonium 210
Methylchrysene
Methylnaphthalenes
Benzo[e]pyrene
Nickel
Arsenic
Cadmium
the Ames Test

How to identify carcinogens?
 Animal testing
 Reliable but slow and expensive
the Ames Test

Ames test: does the agent cause mutations in bacteria?
 Fast, cheap
 Mutagens tested further
 Non-mutagens probably safe
Bruce Ames
the Ames Test

Wild-type Salmonella can make histidine (his+)
 Grows on minimal medium (no histidine)

his- mutant (auxotroph) can’t make histidine
 Can’t grow on minimal medium
Salmonella typhimurium
the Ames Test

Mutation can make a his- cell his+ (reversion)
 Grows on minimal medium
 Low spontaneous mutation rate
no histidine in plate
1010 his–
bacteria
most die; few
spontaneous
his+ mutants
the Ames Test

Mutagen increases frequency of his+ mutation

Test a possible mutagen and see if mutation rate increases
1010 his–
bacteria
mutagen
increased
chance of
mutation
to his+
no histidine in plate