Molecular Biology II Molecular Medicine 1 - Genome Rearrangements

Chromosomal Abnormalities:

Traditionally, only large gains/ losses could be detected (>4Mbp)

Now smaller changes can be detected – molecular cytogenetics and FISH detection
Most abnormalities are due to:
 Broken chromosomes
 Improper recombination
 Mal-segregation during mitosis / meiosis
 Fertilisation & early development errors

e.g. polyploidy


Multiplex FISH (Fluorescent In Situ Hybridisation):

Used to visualise chromosomes

Chromosome Painting:
3 fluorochromes are used in different combinations to give many colours
Chromosome specific probes are used each with a different colour

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If 2 colours are seen on one chromosome then chromosome translocation has taken place
Chromosomal Abnormalities:
 Constitutive abnormality – all cells, occurs early in development e.g. fertilisation
 Somatic abnormality – mosaic
 Numerical - Triploidy (~2% conceptions)
- Tetraploidy (four sets of chromosomes, lethal – unless on X chromosome)
- Trisomy (one extra chromosome, survival rates depend on chromosome)
o Down syndrome (trisomy 21) – can survive longer
- Nullisomy (missing chromosome, embryonic lethal)
 Deletions, inversions, duplications, insertions
 Unequal parental contribution (rare)
o ‘male-driven’ evolution  driven by abnormalities in sperm
o More divisions in sperm formation,  mutations in sperm
 Many diseases and syndromes are involved with chromosomal abnormalities

Small-scale Mutations:
 Nucleotide substitution (synonymous / silent), deletions, insertions
 Protein polymorphisms
 Expression polymorphisms
 Single nucleotide polymorphisms (SNPs)
 Restriction site polymorphisms

1.5% human genome is coding, 3-4% is regulatory

Error rate in DNA replication is ~10-10 before correction
A coding gene is ~1.65kb on average
 1.65 x 10-7 mutations / gene / division
1016 mitotic cycles in a human lifetime, 109 mutations will occur within coding genes in a lifetime
Most will only affect one cell, so inconsequential for the organism (apart from cancer)

Larger-scale Mutations:
 Variable Number Tandem Repeats (VNTR) polymorphisms
o Simple - Rarely in genes or regulatory sequences but can affect expression
- Microsatellites (1-9nt long, overall length ~100)
- Minisatellites (9-10nt long, overall length ~100s)
o Large scale - α-satellite repeats, rRNA
 Transposition repeat polymorphisms
o LINE, Alu, LTR-based

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Often involved in DNA exchanges and instability

Repeated DNA leads to pathogenesis:

Tandem Repeat Mutation Example

Very short repeats in genes Deletion / frameshift Slipped strand miss-pairing (SSM)

Moderate sized intergenic repeats Intra-genic deletion Unequal cross-over

Partial / total gene deletion Unequal cross-over

Large tandem repeats of whole genes Altered gene sequence / gene conversion Allele conversion

Interspersed Repeats Mutation Example

Short direct repeats Deletion SSM / recombination

Interspersed repeats (e.g. Alu) Deletion / duplication UEC / UESCE

Inverted repeats Inversion Intrachromosomal exchange

Active (retro-) transposons Intragenic insertion

Genetic Mechanisms for Sequence Exchange:

Replication slippage / slipped strand miss-pairing

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Equal crossover Unequal crossover

Homologous recombination

A deletion may result in pathological consequences

Recombination / pseudogenes

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Pseudogene (ψ) = 21A

21 hydroxylase = 21B
Complement = C4A/B

21 hydroxylase deficiency causes congenital adrenal hyperplasia

 Defects in steroid synthesis (affects testosterone / progesterone)
 Infant salt wasting (steroids are involved in salt metabolism)
 Infant female virilisation (sex hormone imbalance)

Inversion at inverted repeats

Homology between a region of factor 8 + 2 genes far from this region
A region of DNA containing introns gets flipped exons are now in wrong order
Results in factor 8 deficiency

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Splicing mutations

SD = Splice

Donor
SA = Splice Acceptor

Mutations (deletions) in the 2nd SA or SD will result in a large amount of intron DNA being included
as an exon.

Or can result in an entire exon being missed out. Information is lost.

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A mutation can result in the introduction of an

unwanted splice site.

All of these splicing mutations will result in aberrant transcripts.

Programmed Genomes Re-arrangements:

Gene amplification occurs naturally
 Xenopus rDNA genes
 Egg shell protein (chorion) gene of Drosophila ovarian follicle cells during development
 Tumor cells (oncogenes such as Her-2) – Break-fusion-break model
The target enzymes for anti-cancer drugs such as methotrexate (DHFR) will have hundreds of
extra copies on extra-chromosomal elements in response to treatment (drug resistance).

“Break-fusion-break model” – a cause of genomic instability in cancer cells

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Detailed mechanism needs to be looked up

Eukaryotic Genomes Rearrangement:

Immunoglobulin gene diversity – creates cell differentiation in B-cells
Similar enzymes and process to transposition
Recombination signals/RAG????
Some DNA is lost forever in B-cell development this irreversible damage is programmed into the
cell.

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Transposons:
Mobile genetic elements which can migrate.
Transpose independently = autonomous (have the ability to move independently)

Make up almost all of interspersed repetitive non-coding DNA

Unknown if this DNA is junk or if it has some value
A very small minority are actively transposing

Retro-transposons (replicative or non-replicative)

LINEs SINEs & retrovirus-like LTRs
DNA transposons (conservative transposon – cut & paste)
Mariner element

LINEs and SINEs (e.g. Alu repeats) dominate

Non-LTR Retro-transposons:
More abundant than LTR containing
Most have lost the ability to transpose
Junk DNA? Facilitate exon shuffling. Recombination ability can drive evolution.
Similar to telomere it is unknown where the two originated from
Long Interspersed Nuclear Elements (LINEs) e.g. L1 (might have a role in telomere protection)
100000 copies (15% of human genome)
L1 is found in some genes leading to mutation
 Blood clotting factor VIII
 DMD gene (Duchene Muscular Dystrophy)
 APC or Rb gene – leading to cancer

Retro-transposons:
DNA replication via an RNA intermediate

Autonomous retro-transposons contain

 Gag = core
 Pol = RT/integrase/protease
These proteins facilitate movement
The genetic material is similar to that of retrovirus RNA
They contain an LTR (Long Terminal Repeat)
 Recombination via LTRs is similar to retrovirus
activity

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e.g. Drosophila copia element or Yeast Ty (transposon yeast) element

Retroviruses:
RNA viruses that cause disease
Utilise a similar copying mechanism to retro-transposons
Retroviruses use the host cell machinery to produce proteins in order to package new viruses
It is not know if retroviruses are derived from retro transposons or the other way around

A DNA copy of the RNA genome is generated

within the host cell by reverse transcriptase
LTRs are found at each end
Contains the pol gene as above
(integrase reverse transcriptase protease
RNase H)
Integrated (into host genome) virus = provirus
Uses a tRNA (trp) primer for replication
Can be used for gene therapy

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