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MutagenesisMutagenesis

Definitions: GenotypeDefinitions: Genotype

• Komposisi genetik dari suatu organisme• Mutasi diidentifikasikan dengan penomeran pada gen yang mengalami mutasi atau diberi tanda (-)

e.g. leuB6 or leu-, leuB6 or leu-

leuB6 = mutasi pada nomor 6 pada leuB gene

leu- = mutasi pada posisi yang belum diketahui pada leu gene, (dapat diidentifikasi dari dibutuhkannya suplemen leucine saat tumbuh dalam minimal media).

PhenotypePhenotypeSifat yang dapat terlihat dari suatu host, mis: rpsl (StrR) StrR is merupakan phenotye, dan menunjuk-kan bahwa resisten terhadap Streptomisin

Oleh karena fenotip tidak menjelaskan tentang gen, maka penulisan tidak perlu italik

Sebagai konsensus, penulisan fenotip dan genotip menggunakan 3 huruf.

Mutation, mutants & mutagensMutation, mutants & mutagensMutation • Perubahan basa dalam sekuen DNA (Pd umumnya di dalam suatu gene). • Perubahan ini termasuk : base substitution, addition, re-arrangement or deletionMutant • Organisme yang mengalami mutasi. • Mutatisi tersebut tentunya terjadi dalam gene yang menyebabkan terjadinya perbedaan yang nyata dengan bentuk normal (Wild-Type).

Mutagen• pengaruh fisika atau senyawa kimi yang menyebabkan mutasi.

Mutagenesis

- Proses terjadinya suatu mutasi- Bisa terjadi secara:

(a) Spontan (natural process) terjadi <1 in 108 E. coli cells.

(b)Diinduksi (e.g. dengan mutagen).

Mutasi dapat terjadi dalam satu atau dua hal:

1, Mismatch – terjadi karena kesalahan pada DNA replikasi.

2, perubahan struktur – diinduksi oleh mutagen (physical and chemical agents).

Mismatches Mismatches saat DNA replikasi saat DNA replikasi

5’-ATTGG-3’3’-TAACC-5’

5’-ATGGG-3’3’-TAACC-5’

5’-ATGGG-3’3’-TACCC-5’


Normal

Mutated

Replication

Perubahan struktur Perubahan struktur Mutasi DNA Mutasi DNA

5’-ATXGG-3’3’-TAGCC-5’

5’-ATXGG-3’3’-TAGCC-5’

Normal

5’-ATXGG-3’3’-TAACC-5’


5’-ATCGG-3’3’-TAGCC-5’

Kerusakan

nukleotida

Mutation

Mutation

Tipe mutasiTipe mutasiMutasi pada level DNA1. Point mutation Perubahan satu basa dengan nukleotida yang lain

2 types:Transition – perubahan purine ke purine (A to G, G to A)

atau pirimidin ke pirimidin (C to T, T to C)

Transversion – perubahan purine ke pirimidin atau sebaliknya, e.g. A to C or T, C to A or G

2. Insersi or delesi Penambahan atau pengurangan satu atau lebih base-pairs.

HUNTINGTON DISEASE

CAG : GLUTAMINNORMAL 6 – 31 KOPIPENDERITA 36 -82 KOPI

Tipe mutasiTipe mutasi

3. Inversi Pemotongan sebagian DNA dan kemudian disisipkan

pada tempat yg sama tetapi pada orientasi yg berbeda

5’-ATTGG-3’

3’-TAACC-5’

5’-ATCAG-3’

3’-TAGTC-5’

Mutasi pada level geneMutasi pada level gene

1. Silent mutation

Perubahan basa tidak meyebabkan perubahan pada kodon

5’ ATG GGA GCT CTA TTA ACC TAA 3’ met glyala leu leu thr stop

5’ ATG GGA GCT CTA TTG ACC TAA 3’ met glyala leu leu thr stop

Silent mutation( & transition)

2. Missense mutation Perubahan basa Perubahan kodon


5’ ATG GGA GCT CTA TTT ACC TAA 3’ met glyala leu phe thr stop


Missense mutation( & transversion)

3. Nonsense mutation Perubahan basa menjadi stop kodon


5’ ATG GGA GCT CTA TGA ACC TAA 3’ met glyala leu stop


Nonsense mutation( & transversion)

4. Frameshift mutation Hilangnya satu basa perubahan pada pembacaan


5’ ATG GGG AGC TCT ATT AAC CTA A 3’ met glyser ser ile asn leu


Frameshift mutation

Mutasi pada level organismeMutasi pada level organisme

Lethal mutations – organism dies as a result of an altered (or absent) gene product that lacks function. Could be a conditional lethal.

Mutations which cause only a partial reduction in the function of the gene product

Mutations in genes whose function is not critical to the cell or is compensated by other genes

Silent mutations (no effect)

ReversionReversionThis is the reverse process of mutation and involves a mutant regaining a wild-type phenotype, either through:

1. A back mutation (direct reverse of the mutation)2. A reverse or suppressor mutation (not a direct reverse, but a mutation at a second site generally within the same gene which

suppresses the effect of the first mutation). The mutant is described as a revertant.

A practical example of reversion is the Ames test which is used to test for carcinogens.

What is reversion?What is reversion?

+-

WT, active

+

+

Mutate - to + Mutant, inactive

+-

WT, activeSame site revertant

+-

Mutate + to -

WT, activedifferent site revertant

Ames TestAmes Test

Background spontaneous

reversion

Chemical inducedreversion

Minimal media minus histidine

Test chemical + bacteria

48 hrs

Ames TestAmes Test

Is based on histidine-requiring mutants (his-) of Salmonella typhimurium.

The test measures mutagenicity by an increase in the

frequency of spontaneous reversion of his- to his+

Is used to test: industrial chemicals, food additives, pesticides, hair dyes, cosmetics.

Reduces the need for animal testing.

MutagensMutagens

A spontaneous mutation occurs once in 108 cells.

The mutation rate can be increased by exposing cells to mutagens, which are either chemicals or physical agents such as UV-irradiation.

Both chemicals and physical agents act by causing genetic damage that results in base changes in DNA

Chemical MutagensChemical Mutagens1. Base analogues e.g. 5-BromouracilThis base is incorprated in the same way as thymine,(its analogue of thymine) during DNA replication. However the base exists in 2 different forms (tautomers),one of which (the keto form) base-pairs with adenine, And the other form (the enol form) base-pairs withguanine.

Clearly any base that alters its base-paring is going to bemutagenic.

Causes A-T to G-C changes, which are ? Transitions

(from Microbial genetics by Maloy et al.)

Chemical MutagensChemical Mutagens2. Chemical mutagens e.g nitrous acid and ethyl methane sulphonate.

The chemical reacts with a base such that the chemicallly modified base behaves differently.

Mutagen Base Base change Effect Net effect HNO2 + cytosine uracil C-G to U-A (C to T)HNO2 + adenine hypoxanthine A-T to H-C (A to G)

EMS + guanine alkylated G G-C to G*-T (G to A)

Chemical MutagensChemical Mutagens2. DNA intercalating agents

These agents are planar (flat) in structure and are approximately the same size as a purine-pyrimidine base-pair. In solution they can insert between stacked base-pairs.The best known such agent is ethidium bromide which is used to visualise DNA, as when it is inserted between the stacked base-pairs it fluoresces brightly to allow the DNA to be visualised. Replication of DNA containing intercalating agents is often seen to result in the addition of single bases, which seriously affects the reading frame of the gene.

TransposonsTransposonsThese are mobile elements (pieces of DNA) that move

within the bacterial genome, at between 10-7 and 10-2 events per generation depending on the mobile element. Jumping genes!

The transfer from one location to another is known as transposition. While resembling a recombination event, it is illegitimate recombination as it doesn’t require RecA protein.

If the transposon locates within a gene it will act like an insertion mutation, however, treating with mutagens that cause frameshift mutations, or single additions or deletions did not cause reversion, because of the insertion of the transposons

2 types of transposons2 types of transposons

1. Insertion Sequences or IS elementsThese are the simplest form of transposon and carry inverted terminal repeat sequences (9-41 bp) and a transposase gene. Total size = 768 bp to 6000 bp

The transposase gene recognises the terminal repeats and catalyses the transposition to another loci.

It doesn’t code for any other genes!

IS1 element (768 bp)

TransposaseInverted

repeat

2 types of transposons2 types of transposons2. Composite transposonsThese contain 2 IS elements flanking an antibiotic resistance gene (e.g. resistance to kanamycin,chloramphenicol, tetracycline, fusidic acid).

Antibiotic resistance gene

IS Element IS Element

Transposition involves duplication of Transposition involves duplication of a target sequence (9-41 bp)a target sequence (9-41 bp)

Single-strand break at target sequence

Attachment of transposon

Filling-in and sealing

Target sequence where transposon Target sequence where transposon integrates becomes an inverted repeatintegrates becomes an inverted repeat

5’-AGGCAT-3’3’-TCCGTA-5’

5’-AGGCATATGCCT-3’3’-TCCGTATACGGA-5’

5’-AGGCATATGCCT-3’3’-TCCGTATACGGA-5’

Left end Right end

IS / Tn

Effect of transposonsEffect of transposons

Gene A Gene B Gene CPromoter

Expression of all genes affected

Expression of all genes affected

Gene A only expressed

Site of insertion & effect

Genes A& B only expressed

Transposons have a polar effect, through their insertion, which cannot be reversed by agents that cause insertion or frame-shift mutations.

Transposon mutagenesisTransposon mutagenesisTransposons are widely used to generate mutations in chromosomal and plasmid genes.

While transposition may be rare, the presence of an antibiotic resistance gene allows easy selection.

Moreover insertions at sites close to your gene of interest greatly facilitates strain construction or genetic mapping.

Transposons are the reason why there is such widespread multiple antibiotic resistant bacteria in hospitals, as there is a strong selective environment by indiscriminate use of antibiotics.

BibliographyBibliography

**Microbial Genetics by S.R. Maloy, J.E. Cronan & D. Freifelder

**Major reference

Learning Objectives:

Understand the concepts of genotype, phenotype, gene nomenclature/notation.

Understand what a mutation is, how they arise, and their consequences.

And in the later lecture how damage to DNA is repaired, and maintained.

Definitions: GenotypeDefinitions: Genotype

E. coli strain, HB101. F- (gpt-proA)62 leuB6 glnV44 recA13 ara-14 galK2

lacY1 mtl-1 xyl-5 recA13 rpsl (StrR)

This strain would grow on LB agar plates

And on minimal plates, supplemented with: proline & leucine (N.B. glnV44 is a mutation in a tRNA not a gene involved in glutamine metabolism).

Unable to use galactose, lactose, mannitol or xylose as a carbon source

Types of mutantsTypes of mutants1. Auxotrophic mutantsMutation has inactivated a gene involved in theproduction of an essential metabolite, e.g. leuB6 E. coli with this mutation require leucine to be supplemented to minimal mediaThe WT is prototrophic, does not require supplements.

2. Conditional lethal mutantsMutants that can only survive if cultured under aparticular set of conditions, e.g temperature sensitive Mutations can only grow at permissive temperature (e.g. 30oC) and die at restrictive temperature (e.g. 37oC) .ONLY mutant at one temperature (e.g. 37oC) .

Types of mutantsTypes of mutants

3. Antibiotic resistant mutantsThe target for the antibiotic becomes altered (e.g. Streptomycin resistance due to a change in ribosomal protein S12).

4. Regulatory mutantsHave lost the ability to control expression.For example, constitutive mutants which express genes of the lac operon even in the absence of lactose (e.g. mutation in the lacI repressor).

Types of mutantsTypes of mutants1. Auxotrophic mutantsMutation has inactivated a gene involved in theproduction of an essential metabolite, e.g. leuB6.

E. coli with this mutation require leucine to be supplemented to minimal media, as the mutation is presumably in a gene that makes a protein that is involved with synthesis of leucine.

The WT host with a normal leu gene is prototrophic, as it does not require supplements.

Types of mutantsTypes of mutants2. Conditional lethal mutants

Mutants that can only survive if cultured under aparticular set of conditions, e.g temperature sensitive.

Mutations can only grow at permissive temperature (e.g. 30oC) and die at restrictive temperature (e.g. 37oC) .ONLY mutant at one temperature (e.g. 37oC).

Types of mutantsTypes of mutants3. Antibiotic resistant mutantsThe target for the antibiotic becomes altered (e.g. Streptomycin resistance due to a change in ribosomal protein S12). These can be obtained by plating bacteria oncontaining agar containing streptomycin.

4. Regulatory mutantsHave lost the ability to control expression.For example, constitutive mutants which express genes of the lac operon even in the absence of lactose (e.g. mutation in the lacI repressor).This will be illustrated by lectures on the lac & trp operons.