BI2002

Genes and Evolution

L4,5: mutation

Dr Ken Forbes
Medical Microbiology
 !
OB

Phenotype and genotype

• Phenotype
– observed characteristic
» eg growth

• Genotype
– genetic basis of phenotype
DNA sequence
Phenotype and genotype

• Many different genotypes can have same

phenotype
– one phenotype can be due to many different
genotypes

• Many genotypes do not change the phenotype

Bacteria are good model
systems . . . . .
Bacteria are …….
• Unicellular
• Chromosome
– single
– haploid
• Replicate by binary fission
– daughter cells identical to parent
– clonal
– NO genetic variation from growth cycle
• Simple gene structure
 …….. Gene Expression

Promoter
Operator Gene A Gene B
DNA

rbs AUG STOP AUG STOP

mRNA

protein
Protein A Protein B

• DNA and mRNA sequence of nucleotides

• nt triplet (codon) codes for one amino acid
 Mutants - why use them ?
! !
PENGGUNAAN MUTASI DALAM PENELITIAN OB OB
• Mutations
– make the system defective
– study the changes

• Biochemistry
– metabolism
– biochemical pathways

• Genetics
– more detail on pathways
– control systems
Mutants – in bacteria

• Inability to use a substrate

– sugar
• Auxotrophs
– amino acid
– vitamin
• Resistance
– antibiotics
– phage
– toxic chemicals
Mutants - selection and detection

• Non-selective media
– all cells grow so test individual colonies
» replica plating
• Selective media eg:
– antibiotic present
– absence of amino acid
• Indicator media
– MacConkey agar (Lac+/-)
Mutants – in higher organisms

• Appearance
– Colour (mouse coat)
– Shape (Drosophila wing)
– Size (dog breeds)

• Behaviour
– Inborn errors of metabolism
Why make transgenic animals?

• Find out where a gene is expressed

(i.e. reporter constructs) --> mengetahui lokasi
produk ekspresi gen
• Over express or ectopically express a gene
(i.e. create a dominant gain of function phenotype)
• Knock out a gene
• Genetic mosaic analysis
• Clone a gene by transformation rescue
Types of mutations !
OB
• Point mutations affect a single gene
– nucleotide substitutions
= SNPs (single nucleotide polymorphisms)
– frameshifts

• Larger-scale mutations
Point Mutations: nt (nucleotide) substitutions)
....TAC....
....ATG....
!
OB

TAC
ATG

Transcription
UAC
TYR codon

Translation
Normal
Protein
Wild
type
Point Mutations: nt (nucleotide) substitutions)
....TAC....
....ATG.... !
OB

TAC TAT AAC TAG

ATG ATA TTG ATC
Transcription

UAC UAU AAC UAG

TYR codon TYR codon ASN codon Stop codon
Translation

Normal Normal Faulty Incomplete

Protein Protein Protein Protein
Wild Silent Missense Nonsens
type mutation mutation mutation
Point mutations: frameshift

AUGCUAGCUAGCUUACCUAUUCGA
Met Leu Ala Ser Leu Pro Ile Arg

• Reading frame of nt sequence

– in frame
Point mutations: frameshift !
OB
AUGCUAGCUAGCUUACCUAUUCGA
Met Leu Ala Ser Leu Pro Ile Arg
• in frame

AUGCUACUAGCUUACCUAUUCGA -
• out of frame
Met Leu Leu Ala Tyr Leu Phe

AUGCUACUAGUCUUACCUAUUCGA -
Met Leu Leu Val Leu Pro Ile Arg
• in frame
Larger-scale mutations
!
OB
• Deletions
– removes gene(s)
– may change phenotype
• Insertions
– adds gene(s)
– may change phenotype
• Rearrangements
– re-orders gene(s)
– may not change phenotype
Reverse and Suppressor mutations

• Forward mutation
WT phenotype  mutant phenotype
– wt sequence  mutant sequence

• Reversion
mutant phenotype  WT phenotype

– Reverse (back) mutation

» mutant sequence  wt sequence
– Suppressor mutation
» mutant sequence  more mutations
Forward genetics: !
OB

• A mutant phenotype is identified and and we like

to know what gene is mutated
• Based on the identity of the gene and the mutant
genotype we can then infer the function of that
gene, or at the very least what process the gene
is involved in
Forward genetics: !
OB

An example is the REF8 gene of Arabidopsis. The

ref8 mutant, which has the mutated copy of the
REF8 gene (Franke et al., 2002), accumulates a
particular type of lignin precursor, the H-unit. We
can therefore infer that the FUNCTIONAL REF8
gene is involved in preventing the accumulation of
H-units.
Reverse genetics: !
OB
• deals with the situation where we have a gene
sequence, but we do not know the function of the
gene
• we try to identify mutations in the gene of interest,
and evaluate the organism/cell that carry the
mutations
• Based on the mutant phenotype we can then infer a
function of the gene.
• This approach tends to work best with mutations as a
result of insertions, combined with PCR.
Mutagenesis

Spontaneous mutation rates

• Measuring frequency of mutation
– Need large populations
– Often use special techniques

• Mutation rate =
Ratio in a population of

( )
Number of mutants
Number of WTs
• “mutants/cell/generation”
 Mutagenesis

Mutation rates
• Spontaneous mutations tend to be rare!
– in bacteria typically 10-6 mutants/cell/generation

• Used in
– population genetics
– evolutionary studies
– measuring effects of mutagens
Mutagenesis
!
OB

Mutagenesis
Spontaneous mutations
• Induced Mutations (mutagens)
– chemical
– radiation
Spontaneous mutations
KARAKTERISTIK MUTASI SPONTAN !
OB
• Mutation is a random event
• Mutations occur independently of a selective
(dis) advantage to host
• Each gene mutates at a characteristic rate
– Probability of mutation in a particular gene
• Each type of mutation occurs at a characteristic
rate
Mutagens

Base analogues
!
OB

• Molecule similar to one of the four DNA bases

– can ONLY be incorporated into DNA at replication
– can pair with a normal base

• Analogue occasionally mis-pairs with other bases

– nt change (mutation) will then occur during DNA
replication
5-bromouracil: a base analogue
• analogue of thymine so pairs with adenine
• conformational change of 5-BU leads to
pairing with guanine

. . . A . . .
. . . T . . .
!
CLB
Incorporate 5-Bu
. . . A . . .
. . . Bu. . .
DNA replication
. . . A . . . . . . G . . .
. . . T . . . . . . Bu. . .

. . . G . . .
. . . C . . .
Intercalating chemicals
• Planar, ringed molecules the size of a bp
– acridine, ethidium bromide
• Intercalate into dsDNA between bp’s
• At DNA replication get nt added (or deleted)
in daughter strand
• Frameshift mutations in coding sequence
Radiation: ultraviolet
• UV energy absorbed by base
• Chemical modification of base
• Adjacent pyrimidines covalently bond
– Pyrimidine dimers
• DNA helix distorted
– Replication & transcription blocked
Radiation: ionising
• ,  particles, ,  rays
• Free radicals formed
• React with and damage DNA
– ssDNA breaks
– dsDNA breaks hard to repair
– nt substitutions
• Dose  mutation rate