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Dosen : Cico Jhon Karunia Simamora, SP. MSi.

Prodi Ilmu dan Teknologi Pangan

Universitas Tanjungpura

Microbial Genetic

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SIFAT KIMIA DAN MOLEKUL

PENYUSUN DNA

Merupakan molekul yang sangat panjang
tersusun atas 4 jenis molekul NUKLEOTIDA
Nukleotida terdiri dari 3 komponen:

Fosfat, Gula Pentosa (Deoksiribosa), dan Basa Nitrogen

BASA NITROGEN terdiri dari 2 kelompok:

Kelompok Purin : Adenin (A) dan Guanin (G)

Kelompok Pirimidin : Citosin (C) dan Timin (T)

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Adenin (A) berpasangan dengan Timin (T)

dengan dua ikatan hidrogen, dan

Guanin (G) berpasangan dengan Citosin (C)

dengan tiga ikatan hidrogen

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Model DNA UTAS GANDA

(Watson & Crick, 1953)

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STRUKTUR DNA

terdiri dari dua rantai Polinukleotida yang bersifat Antiparalel

(5’P - 3’OH // 3’OH - 5’P)

antar rantai nukleotida berikatan pada basa-N secara

Komplementer (A=T) dan (G≡C)

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Ekspresi Gen pada Prokaryot

Dan Eukaryot

Gen ?

Fragmen DNA yang menyandikan

Suatu protein atau enzim.

~Prokaryot: transkripsi & translasi terja-

di di dalam sitoplasma.

~Eukaryot:

transkripsi di dalam inti sel

translasi di dalam sitoplasma

~Ekson dan intron ditranskrip memben-

tuk RNA utama (Pre-mRNA), selanjut

nya terjadi proses intron splicing.

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Mutations and Mutants

Mutation: genetically inheritable change in one or more genes (during replication)

Change in DNA sequence

Often leads to change in function of gene product

Wild-type (wt): normal (not mutated)
Mutant: organism that has a change (mutation) in its genome

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Point Mutation

A point mutation is a change of a single nucleotide to one of the other three possible nucleotides

Transition
purine replaces purine
A G or G A
pyrimidine replaces pyrimidine
C T or T C

Transversion

purine replaces pyrimidine or
pyrimidine replaces purine
A or G T or C
T or C A or G

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Missense Mutation

A point mutation that exchanges one codon for another causing substitution of an amino acid

Missense mutations may affect protein function severely, mildly or not at all.
Hemoglobin mutation
glutamic acid valine causes sickle cell anemia

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Nonsense Mutation

A point mutation changing a codon for an amino acid into a stop codon (UAA, UAG or UGA).

Premature stop codons create truncated proteins.

Truncated proteins are often nonfunctional.

Some truncations have dominant effects due to interference with normal functions.

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Insertion or Deletion

Mutations

The genetic code is read in triplet nucleotides during translation.

Addition or subtraction of nucleotides not in multiples of three lead to a change in the reading frame used for translation. Amino acids after that point are different, a phenomenon called a frameshift.

Addition or subtraction of nucleotides in multiples of three leads to addition or subtraction of entire amino acids but not a change in the reading frame.

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Not all Mutations Impact Protein Function

Silent mutations are mutations that do not alter the

amino acid encoded.

AAA and AAG both encode the amino acid lysine.

A mutation from AAA to AAG in a gene alters the DNA sequence but protein sequence remains unchanged.

These codons are called synonymous codons.

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Not all mutations impact protein function

Missense mutations are those that alter the encoded amino acid to another amino acid.

The alteration creates a nonsynonymous codon.

Some nonsynonymous mutations are conservative;

chemically similar amino acid may not alter function

The impact of a missense mutation is not predictable from protein sequence alone.

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Reparasi mutasi

mismatch repair mengenali nukleotida dengan padanan yang tidak sesuai pada utas baru kemudian dipotong, dikeluarkan, diperbaiki dengan bantuan gen mut, eksonuklease, DNA pol III

excision repair memperbaiki DNA yang rusak dengan menyingkirkan nukleotida dan mensintesis kembali dengan bantuan endo- dan eksonuklease, polimerase dan ligase

fotoreaktivasi dengan bantuan enzim khusus (fotoliase) yang digiatkan oleh cahaya kasatmata memotong ikatan kovalen diantara dimer-dimer yang terbentuk akibat terkenai sinar UV dan memulihkan aktivitas normal sel-sel yang rusak.

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What is a Plasmid

DNA extra chromosome, small circular
Autonomous replication
Variable number of copies and size
Non essential for the basic operation of the cell
Contain antibiotic resistance gene or the others
Plasmids that confer conjugation (F plasmids)
As cloning vectors (multicopy)

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Plasmid

Chromosome

Escherichia coli

Cell

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Figure . Physical map of the complete genome

sequence of Shinorhizobium meliloti

Mega Plasmid

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Fig . Map of plasmid expression vector pET-15b

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3 Ways of DNA/Gene Transfer

in Bacteria

1. conjugation – plasmid/DNA transfer via physical contact

2. transformation – absorb DNA from environment

3. transduction – bacteriophage-mediated DNA transfer

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In 1946, Lederberg and Tatum showed that two different strains of bacteria with different growth requirements could exchange genes
Lederberg and Tatum surmised that the bacterial cells must interact with each other - the process is now known as conjugation

Genetic information can be transferred between bacteria

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BACTERIAL CONJUGATION

fertility factor – F plasmid
F⁺ donor & F^– recipient strains
F⁺ x F^– 🠞 both F⁺
unidirectional rolling circle plasmid replication
F DNA transferred through a pore in the pilus

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BACTERIAL CONJUGATION

the F plasmid can integrate into the host chromosome
generates a high frequency recombinant strain ... Hfr

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Bacterial Transformation

1. DNA originating from donor bacteria

Cell death lysis release of DNA

2. Uptake of DNA by recipient bacteria

Prior to uptake the cell must be competent

3. Integration of transforming DNA

Competence - ability of cell to take up DNA

 changes in cell wall

 formation/activation of DNA receptor proteins

 natural competence eg. Bacillus, Neisseria spp.

 artificially induced competence -

CaCl₂/cold treatment - low efficiency,

Heat shock / Electrophoration

used routinely in DNA cloning eg. E. coli

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Bacterial Transformation

1. Lysis

3. Recombination

and exchange

Cell B: Recipient

2. DNA

Uptake

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Transformation

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Bacteriophage and Transduction

Bacteriophage

viruses that infect prokaryotic cells

obligate intracellular parasites

two types:

I) Lytic Phage- replicate and subsequently lyse� host cell

eg. phage T4 and T7

(involved in generalised transduction)

Transduction –

bacteriophage-mediated gene transfer

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II) Temperate phage

phage DNA is integrated into the host � chromosome (prophage), where it can be� stably maintained.

phage DNA can be excised, and the phage� can enter the lytic cycle

eg. phage lambda (λ) and P2

(involved in specialised transduction)

Bacteriophage and Transduction (cont.)

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Generalised Transduction

1. Attachment

of phage

2. Insertion

of phage

DNA into cell

3. Replication of

phage DNA

and phage

packaging

Transducing phage

Lytic phage

4. Cell lysis and

release of lytic and

transducing phages

Lytic

cycle

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Generalised transduction (cont.)

5. Attachment of

transducing

phage to new cell

6. Insertion of chromosomal

DNA fragment into cell

7. Homologous exchange

of incoming DNA with

chromosomal of recipient

8. Transduced cell

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Specialised Transduction

1. Attachment

of phage

2. Insertion of phage

DNA into cell

3. Integration of phage

DNA into chromosome

prophage

Prophage induction leading to

excision of phage from chromosome

chromosome

aberrant

excision

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Cell containing

integrated prophage

5. Attachment

of phage

particles

to new cells

4, Synthesis of phage coats,� phage excision packaging of� DNA into phage coats, cell lysis

Normal

phage

Defective

transducing

phage

Specialised Transduction (cont.)

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Transposable elements in

prokaryotes:

Insertion sequence (IS)

Transposons (Tn)

Bacteriophage Mu

1902-1992

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Insertion sequence (IS) elements:

Simplest type of transposable element found in bacterial chromosomes and plasmids.

Encode only genes for mobilization and insertion.

Range in size from 768 bp to 5 kb.

IS1 first identified in E. coli’s glactose operon is 768 bp long and is present with 4-19 copies in the E. coli chromosome.

Ends of all known IS elements show inverted terminal repeats (ITRs).

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Fig. Integrated IS in the chromosomal DNA

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Transposons (Tn):

Genetic element which can transpose from one site to another site in the genome

Similar to IS elements but are more complex

structurally and carry additional genes.

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Figure . Map of pUTmini-Tn5Km1 (A), and transposon Mini-Tn5Km1 (B)

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Figure Molecular mechanism of transposon mutagenesis

MSGM agar plate

MSGM

Magnetospirillum magneticum AMB-1

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Molecular cloning

Molecular cloning can be divided into several steps:

Isolation and fragmentation of the source DNA.

This can be total genomic DNA,

DNA synthesized from RNA by reverse transcriptase,

DNA synthesized by PCR.

Digestion with restriction enzyme~~genome

Joining the DNA fragments to a cloning vector.

Ligase

Sticky end

Blunt end

Introduction and maintainance of recombinant DNA

in a host strain.

Method: Transformation

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Molecular cloning

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“Insertional inactivation”

pBR322 represents an early

generation of cloning vector

constructed in vitro.

TcR TcS

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ORF38

(kDa)

(A) SDS=PAGE. (B) Western Botting

M. Protein Marker (in kDa)

1. Total protein of uninduced cell

2. Total protein of induced cell

3. Soluble lysate of protein ORF38

Purified of protein ORF38
Western Blot of purified protein

RM. Protein Rainbow Marker

ORF38 (~1.5 kb)