DNA recombination & stuff

Horizontal gene transfer from bacterium to bacterium

1. Transformation: Free external GMat uptaken

2. Conjugation: GMat injected (direct contact)

3. Transduction: GMat transverred by virus

Transformation: A cell uptakes external DNA and incorporates it in its own DNA. The foreign DNA enters through the cell membrane, when it's vulnerable (competence state). That happens during cell wal synthesis, or induced with CaCl2, or electroporesis, so it only depends on the recipient bacterium amd the surrounding medium. Transformation in nonbacterial cells often relates to cancer, so it's called transfection.

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Conjugation: One bacterium transfers genetic material to another via direct contact. One bacterium is the donor and the other the recepient of the genetic material. The donor bacterium carries the F-factor (fertility factor) DNA sequence. F-factor allows the donor to produce a thin, tubelike structure called a pilus. Pili inject the recipient, and bring the two bacteria together. The donor transfers genetic material to the recipien, usually a plasmid, a circular piece of DNA. Plasmids often give the recipient a genetic advantage, like antibiotic resistance genes.

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Transduction: GMat transfer via virus! The new synth-ed coat of the virus some times encapsulates host DNA, which it transfers to the next bacteria, sometimes instead its own viral, so you get 'transducing phage particle' which injects but doesn't infect rather gifts organism-compatible DNA for homologus recombination. Co-transduction markers --> phylogenetic distance/ compatibility (whatever). Today we pack desired GMat in viral coats to GModify eukariotes and prokaryotes (transfection).

Transduction from lysogenic cycle = specialized transduction. When viral DNA is cut out incorrectly from host material -> packed -> released it can give infactive and defective transducing particles.

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Integrase (enzyme): snaps phage-λ DNA to host DNA --> site-specific recombination

Tempered phages: non-lethal, long term stay in vell, unlike virulent phages

Mobile DNA = Transposons (RNA-derived transponsons = retrotransposons)

Give short, repetitive sequences at insertion sites. Retrotransposons are plentiful in higher eukariotes -> give most of repetitive DNA sequences in main DNA.

Non viral retrotransposons:

Long INterspersed ElementS (LINES):

Short INterspersed ElementS (SINES):

most frequent Alu sequence, 300bp, 5% of human DNA

Retrotransposons fixed in coding sequences can give genetic diseases

Bacterial transposons = 'jumping genes' no permanent location.

- - -

- Transposition doesn't need much DNA homology

- DNA synthesis of 3-12 bp at integration site

- Reorganization -> homologous recombination can happen (deletion or inversion)

- Transposons near/inside active gene -

- Inactivate disrupting code sequence, activate neighbor gene (promoter of transcriptor activator), deletions & inversions

In bacteria:

Class I transposons: 1 /million /generation:

Insertion sequence includes transposase gene,

between two short inverted repeats (15-15bp)

Class II transposons : 1 /million /generation:

Short repetitive sequences + protein coding gene (antibiotics)

+ transposase gene + resolvase gene.

Class III transposons:

Small bacteriophages: A gene (transposase), B gene (accelerates transposition), Several specific phage genes (structural proteins, enzymes). (Phage μ)

Transposase (enzyme): Duplicates insertion region of host (5-9bp), cleaves target sequence asymmetrically, gaps need filled and ligated --> direct repeats at the flanks.

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Retroviruses & eukariotes

Vertebrate retroviruses

Replication of DNA with RNA intermediate (reverse transcriptase)

3 structural genes

gag: the viral core (a glycosaminoglycan)

pol: reverse transcriptase

env: viral coat glycoprotein

in-between those 3 genes:

LTRs: Long Terminal Repeats: 240-1500bp, contain promoter

[ provirus = integrated in host DNA viral genome ]

oncogenic genes -> oncogenic products have protein kinase activity <- forms="" mutated="" normal="" of="" proteins="" regulatory="" span="">

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Gene amplification

natural or under metabolic stress, or artificial

not understood, homologus recombination -> copy of genes 4 desired function? -> over different generations

example

Overproduction target enzyme, dihydrofolate reductase (DHFR) ->

Resistance against dihydrofolate reductase inhibitor, methotrexate (used in the treatment of leukemia).

Revolutionized chemotherapy: make it shorter, so no time to generate resistance

The resistance against dihydrofolate reductase inhibitor, methotrexate (used in the treatment of leukemia).

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Assembly of antibody gene

Antigene: foreign macromolecule

-> Stimulates growth of immunocyte (stem cell) ->

-> makes unique cell line of identical cells ->

--> produce one specific antibody

(B-lymphocyte = differentiated cell).

Humans can produce 107 different antibodies.

Different antibodies can't be coded by individual genes. Too much info needed!

Antibody variety bcz of

Recombination of exons: DNA 'library of exons' that code for parts of the antibody.

Stem cell -> differentiates to antibody-producing cell (different exon combinations)

Somatic mutations: Antibody genes mutate on certain locations at high rates. The mutations are not inheritated over generations! bcz they are in somatic cells, not gonads.

Structure: IgG - 2 heavy (H) + 2 light (L) polypeptide chains, intra and intermolecular S-S bridges

- Constant (C) and variable (V) parts within each polypeptide chain - 2 antigene-binding locations (continue and discontinue epitopes on the antigene)

- Hypervariable loops

Classes and subclasses of antibodies:

IgM/ IgG/ IgA/ IgD/ IgE

Class, determined by constant part of L and H chain

Three chromosomes contain the gene segments for antibody chains.

Mouse: Chromosome 12 for H chain, chromosomes 6 and 16 for L chain with differenf Constant segments.

Mice (and humans) are diploid, so, 4 chromosome parts can be chosen for L chain, and 2 parts for the H chain.

Gene expression: allele exclusion! -->

Only one of both alleles (on the homologous chromosomes) is activated.

This is quite unique since normally both the maternal and paternal genes come to expression. The only known exception is the X chromosome in women.

The different chromosome parts: For the L chain: V/J/C segments For the H chain: V/J/D and C segments

Via transposition, different potential combinations of V, J, D and C segments are in every B cell = variety of B cells, each with own antibody of IgM or IgD class -> present antibodies on their cell membrane.

The choice between IgM or IgD depends on different splicing products of the RNA transcript.

Tissue of the immune system:

milt and lymph nodes

In primary immune response, cells with best fitting variable part for the circulating antigene are selected -> grow and differentiate (memory cells, antibody producing cells).

At secondary response = long exposure to the same antigen, class changes happen by selection of other constant parts on DNA level:

IgM (Cμ), IgD (Cδ)→ IgG (Cγ), IgE (Cε), IgA (Cα).

Overall diversity is result of:

L chain: • 300 V with 4 J x 2.5 (cutting and splicing between V and J can occur at 4 different sites = 3000) • The last step in the assembly of the L chain is connection of C and J segments. Happes in RNA not DNA level

H chain: analogous between V/J/D and C =5000

Total diversity: 3000x5000=1.5 10^7

!!! Starting from a limited amount of DNA in the stem cell an enormous diversity can be created in the antibody producing cell !!!

Additional diversity bcz of: • Different V segements can change small pieces of DNA (recombination) • Class changes can occur at repetitive exposure to the same antigene • The V sequence in the producing cell are exposed to a high degree of mutation (somatic mutation) at the ‘hot spots’. This causes a fine tuning of the antibody on the antigene.

Total amount of possible antibodies becomes countless.