Session 1: Basic Genetics Flashcards
Bases - Purines/ Pyrimidines
Purines have 2 interlocked rings A and G
Pyrmidines have a single ring C and T
Number of hydrogen bonds?
A-T
G-C
G-C 3 hydrogen bonds
A-T 2 hydrogen bonds
Why is RNA more unstable?
Additional hydroxyl at 2’ position
Nucleosome components
2 each of H2A, H2B, H3 and H4
Linker DNA histone
H1
Euchromatin is…
Extended conformation
Transcriptionally active
Weak binding of H1 histones
Acetylation of nucleosome histones
Heterochromatin is…
Condensed
Not expressed
Tight H1 binding
cis-acting splicing mutations
Within consensus donor/acceptor
Branch point mutations
Disruption of cis-elements e.g. ESEs, binding of SR’s/hnRNPs
Major spliceosome molecules
U1, U2, U4, U5 and U6
Minor spliceosome molecules
U11, U12, U4atac, U5 and U6atac
Chromosome regions commonly showing variation in constitutive heterochromatin
1qh
9qh
16qh
Yqh
9q heterochromatin Inversions
inv(9)(p11q12) third heterochromatin in p arm (10%)
inv(9)(p11q13) all heterochromatin in p arm (0.6%)
Mutations - mechanisms
- DNA damage
- endogenous - internal chem events ( depurination, deanimation oxaditive damage)
- exogenous - enviromental agents
( mutagenic chemicals, UV, ionizing radiation) - Deficiencies in DNA replication/ recombination
- uncorrected errors 1×10-4 - 10-6 mts/ gamete for given gene - Defects in DNA repair
DNA repair mechanisms
- BER base excision repair
- NER nt excision repair
- MMR mismatch repair
- HR holomologous recombination repair
- NHEJ non-homologous end joining
Doxorubicin - how does it work?
Chemotherapeutic agent used in treatm of various cancers inc breast, bladder, ALL
Inhibits Topoisomerase II -> stops DNA replication, prevent further cell division > cell apoptosis
End-replication problem
Telomerase - reversed transcriptase (RNA dependent DNA polymerase);
2 subunits :
-TERT - protein subunit and
-TERC -RNA subunit with tandem reapeat seq complim to telemere repeats; this provides template to extend the telomere
Chromatine proteins in disease
- CTCF - chromatin loop eg. Silver Russell, Beckwith-Wiedemann Syndrome
- Cohesin - pairing of sister chromatids eg. Cornelia de Lange, Roberts Syndrome
- C-MYC - maintain euchromatine open eg. various tumours
- MLL - inv in early develop and hematopoiesis eg acute leukemias, poor prognosis
- MECP2 Rett Syndrome
- CREBBP Rubinstain -Taybi Syndrome
Telomere function
- Maintain structural integrity – if lost the chromosome end is unstable (becomes ‘sticky’);
- Prevents shortening of the chromosomes at each round of cell division.
- help to establish the 3-D architecture of the nucleus and aid chromosome pairing.
Telomeropathies
· Cri du Chat syndrome (CdCS)
· Dyskeratosis congenital (DC)
· Anaplastic anaemia
Splicing regulation
Regulation
in tissue (cell chemistry),
context (interaction with other trans-acting elements),
timing (cell cycle)
Splicing regulatory elements: ESS, ESE, ISS, ISE - can be cis-/trans- acting
Alternative splicing - types
- exon skipping
- altern 5’ splice site
- altern 3’ splice site
- intron retention
- mutually exclusive exons
Altern splice proteins - examples
- WT1 - 4 isoforms: +/- KTS isoforms (3AA in ex9), +/-17aa (in ex 5)
- Calcitonin (CGRP) - tissue specific splicing
ex 1-4 - thyroid C cells
ex 1-3, 5-6 - neurons - Tau (MAPT) - 3R/4R tau isoforms (alter splicing of ex 10) > FTDP-17
Mutations within consensus splice sites account for (%)
10% of all disease causing mutations
Splicing in disease - mechanisms
- Disruption of splicing element (within +/-2bp, branch point, cis- elements) eg WT1 -KTS isoform, 3bp del in ex4 MLH1 > HNPCC
- Toxic RNA - ^the stability of mRNA-RBP complex> repeat expansion eg DM1 CTG, DM2 CCTG
- Mt affecting splicing factors eg TDP-43 (ALS), RBM20 (DCM)
Founder mutation in FamDys
IKBKAP (ELP1) c.2204+6T>C (skipping of ex 20)
Therapies targeting aberant splicing
- GENE THERAPIES:
- ASO targeting splicing signals ( to enhance/repress splicing) eg
DMD - ASO for skipping ex 51 (Enterlipsen)
SMA- ASO to promote SMN2 ex 7 inclusion (Spinraza)
- ASO/ A/b targeting mutant isoform transcripts for degradation
DM1 ARSE for (CUG)n repeat in RNA DMPK
- SMaRT - post transcriptional correction of mRNA seq - molecules that promote/inhibit the activity of trans-acting factors that regulate alter splicing
mRNA surveillance mechanisms
- Nonsense Mediated mRNA decay pathway (NMD) - targeting transcripts containing premature termination codons (PTCs)
- Nonstop Mediated mRNA decay pathways (NSD) - mRNA transcripts which lack a stop codon
- No-go Mediated mRNA decay pathway (NGD) - mRNA transcripts on which ribosomes have stalled (e.g. due to secondary structures)
NMD
predicted to occur if the PTC is upstream of the 3’ most 50-55 nucleotides of the penultimate exon (last exon-exon junction).
Exceptions to NMD rule
- T cell receptor gene rearrangements. Transcripts degraded by NMD despite having a PTC located within the last 50 nucleotides of the penultimate exon.
- a long 3’ UTR can also target a transcript for NMD eg COL10A1 (mutations cause metaphyseal chondroplasia, type Schmid-MCDS). Mutation hotspot in large final exon 3- trigger NMD but deletion of conserved regions of the 3’UTR protects transcripts (with PTCs) from NMD
- β-thalassaemia- transcripts containing nonsense mutations in 5’ region of exon 1 β-globin escape NMD
NMD and disease
- β-thalassaemia - premature stop in 1st or 2nd exon of three-exon β-globin gene.
- DMD with disrupted reading frame
- Marfan syndrome (FBN1) - dominant neg effect (nonsense mutations> low levels of mutant mRNA = mild phenotype )
- Cancer eg BRCA1, TP53
- ALS (FUS)
MELAS - most freq variant
m.3234A>G MT- TL1 80% cases
MERRF most freq variant
m.8344A>G MT-TK
80 % cases
At which stage of cell cycle recombination occurs
Pachytene - 3rd stage of prophase of meiosis I
Decribe paracentric and pericentric inversion
PARACENTRIC - chr break and inv doesn’t include the cenromere
PERICENTRIC - includes centromere
Homotrisomy
Occurence of more than one child with trisomy in the same family
