Session 1 Flashcards
Outline the stages of the cell cycle
Interphase:
G0 - cell cycle arrest
G1 - cellcular components duplicated
S - Chromosome duplication
G2 - Error checking
Mitosis:
Prophase - Chromosome condense and spindle fibres appear
Prometaphase - spindle fibres attach to chromosomes
Metaphase - Chromosomes line up at metaphase plate
Anaphase - Sister chromatids split to opposite poles
Telophase - Nuclear membranes reform and chromosomes decondense. Loss of spindle fibres
Cytokinesis - cytoplasm divides and 2 daughter cells produced.
What are the three main cell cycle check points?
G1/S - Cell growth enables formation of the CDK4/6-cyclin D. Phosphorylates retinoblastoma protein. Relieves inhibition of E2F transcription factor. Cyclin E now expressed, binds to CDK2
G2/M - CDK1 is activated by phosphorylation and de-phosphorylation of specific amnio acid residues by Cyclin-Activating Kinase (CAK), as well as the inhibition of the wee1 protein (which inhibits CDK1). Enables CDK1-cyclin B formation (aka MPF)
M/Anaphase - Anaphase-promoting complex (APC) activated. Degrades cyclin B = MPF disassembly. Relieves inhibition of ‘separase’ which cuts cohesin. Sister chromatid separation = anaphase entry
How is the cell cycle manipulated for metaphase spreads?
Mitogens - used to induce division of resting cells:(PHA, pokeweed, concanavilin A)
Synchronisation - Inhibitors block cell cycle during S phase by slowing/stopping DNA synthesis (FudR/uridine, Thymidine)
Block released after 16-22h - cells continue through G2 together
Mitotic arrestants - stop division during mitosis (colchicine/Colcemid®). Prevents spindle fibre apparatus formation. Stops cell at metaphase
When does recombination occur?
Prophase 1
What can occur due to non-allelic homologous recombination?
Loss of regions of chromosome - from single gene to multiple genes to entire regions of chromosomes
What are the three subtypes of Eukaryotic chromosomes?
Metacentric: p and q arms are of roughly equal length (e.g. 1 and 3, 16-18)
Submetacentric: the arms are of unequal length (e.g. 2 and 6-12)
Acrocentric: p arm is very short but still present
What are the functions of centromeres?
essential for accurate chromosome segregation during cell division as it provides the foundation for assembly of the kinetochore which attaches to the spindle
Outline centromere structure
Composed of constitutive heterochromatin of families of repetitive satellite DNA - tandem repeats of 171bp AT rich DNA named alpha satellites, that extend for several Mb and make up 3% of the genome.
Around 20 proteins associate to the centromere and exist in two groups on the basis of their spatial positioning throughout the cycle: 1) The first class comprises proteins that are constitutively associated with the centromere such as CENPA, CENPB and CENPC, which are thought to have structural roles in kinetochore formation. 2) The second class known as passenger proteins associate with the centromere transiently during the cell cycle and comprises proteins with diverse roles in cell division such as spindle capture, metaphase to anaphase transition, and sister chromatid cohesion
How does CENPA maintain the centromere position?
CENP-A is a histone H3 variant and it forms a unique complex with other histones (H2A, H2B, and H4) which is only found at the centromere with specific epigenetic mark
Name 3 “diseases” are associated with centromere dysfunction?
Premature centromere division (PCD) - age-dependent phenomenon occurring in women, characterised by rod-shaped X chromosome(s) though to be cause of age-dependent increase of X chromosome aneuploidy. no phenotype
Premature chromatid separation (PCS) - consists of separate and splayed chromatids with discernible centromeres and involves all or most chromosomes of a metaphase. It is found in up to 2% of metaphases in cultured lymphocytes from approximately 40% of normal individuals. no phenotype but maybe increased aneuploidy in offspring/reduced fertility
Roberts syndrome - Autosomal recessive breakage syndrome. ESCO2 encodes Acetyltransferase needed for sister chromatid cohesion in S phase. Metaphase spreads show extensive PCS. Phenotype includes pre/post growth retardation, Limb malformations (reduction), craniofacial (microcephally, clefting), intellectual disability and renal and cardiac abnormalities.
What is the kinetochore?
Large protein complex which assembles on centromere and acts as point of attachment for spindle fibres
What are telomeres?
Highly conserved gene-poor, DNA-protein complexes at end of chromosomes
What is the function of telomerase?
Complex made of TERT and TERC. TERC is RNA subunit used by TERT as template to extend telomeric sequence and maintain telomeres
What is the nucleolus? And what regions organise it? How can it be visualised?
Site of ribosomal RNA (rRNA) transcription, pre-rRNA processing and ribosome subunit assembly
nucleolar organising regions (NOR) located on the short arms of the acrocentric chromosomes
Silver nitrate staining if active
What are the three phases of DNA replication?
Initiation - DNA replication initiates at the origins of replication recognised by the origins recognition complex (ORC).. Topoisomerases create a nick in a single DNA strand to be unwound by Helicases. This releases the tension holding the DNA helix in coils and supercoils and allows the double helix 3’ end of the RNA primers followed by the Polymerase.
Elongation - primers are removed and replaced with new DNA nucleotides and the backbone is sealed by DNA ligase. The two replication forks are created in opposite directions
Termination - two replication forks meet each other and they are dismantled and the two ends are joined by DNA ligase
How do the leading and lagging strands differ?
Leading strand - polymerase moves 5’ to 3’ continuously
Lagging strand - Cannot be continuous due to 5’ to 3’ rule. Polymerase elongates a short stretch of DNA and then moves to a new primer while the helicase moves along the DNA. Synthesis is discontinuous. The short fragments are called Okazaki fragments. Replicative Polymerase is replaced by a different polymerase which has an exonuclease activity to degrade the RNA primer and synthetize DNA. A DNA Ligase then joins the two sections of DNA.
What is the overall error rate per cell division? How is it improved?
1 in 10^3
Proofreading by polymerase and MMR reduces to 1 in 10^9
Name a genetic disease which arises due to variants in aspects of DNA replication?
Origin of replication defect - Meier-Gorlin syndrome
Helicase defect - Bloom Syndrome
Polymerase defect - Hutchinson-Gilford progeria syndrome
Telomerase defect: Dyskeratosis congenita
What families of DNA polymerase exist?
Family B = high-fidelity, DNA polymerases for nuclear DNA
Family A = DNA polymerase γ (gamma) which replicates mitochondrial DNA
What is the structure of DNA?
Linear sugar and phosphate backbone with Carbon 5’ bound to Carbon 3’ of next sugar by phosphdiester bond.
Bases bound to Carbon 1 of sugar molecular
Two strands run anti-parallel to each other with base pairing between Adenine and Thymine (2 hydrogen bonds) and Guanine and Cytosine (3 hydrogen bonds) to form double helix.
What is the structure of RNA?
Single stranded molecule with additional hydroxyl group at the 2’ position which makes it more unstable
What is a histone?
Lysine and arginine rich +vely charged proteins which an affinity for -vely charged DNA
What is a nucleosome?
147bp of 2nm DNA helix coiled in less than two turns around a central core of 8 histone proteins (x2 each of H2A, H2B, H3 & H4)
How is DNA packaged further?
Nucleosomes are separated by stretches of linker DNA which bind H1.
H1 facilitates further packaging into a 30nm thick chromatin fibre made of nucleosome spiral with 6-8 nucleosome per turn
The chromatin forms loops and condenses further to form chromosome
What are the two main levels of chromatin compaction observed in interphase?
Euchromatin - “open” with weak binding of H1 histones and acetylation of the 4 nucleosomal histones
Heterochromatin - highly condensed throughout cell cycle associated with tight H1 histone binding. Can be Constitutive (generally inactive - largely of repetitive DNA) or Facultative (varies)
What are the three main histone post translational modifications?
methylation, acetylation, phosphorylation
What is the effect of histone methylation?
Methylation - commonly on lysines and arginine residues of N-terminal tails which protrude from nucleosomes. Effect depends on which residue is modified due to effect on basicity/hydrophobicity of histones and their affinity with certain proteins . 1. Lysine methylation can be involved in both transcription repression, 2. Arginine methylation has similarly been implicated in both transcription repression
What is the effect of histone acetylation?
Occurs on lysines and almost always associated with activation of transcription. Acetyl group on a lysine neutralises its positive charge and weakens interactions between histones and DNA, de-stablising chromatin architecture. Mediates by HATs and HDACs
What is the effect of histone phosphorylation?
Occurs on Serines, Threonines and Tyrosines. Addition of phosphate to amino acid adds significant negative charge to the histone, influencing chromatin structure. Phosphorylation of H3S10 during mitosis occurs genome-wide - associated with chromatin condensing
What is the mechanism of disease in Rett syndrome?
LoF variants in MECP2
MECP2 binds methylated DNA and recruits HDACS and HMT to respress expression through histone modification. LoF variants lead to activation of genes normally repressed. (mainly in verve cells for phenotype)
What are the three main mechanisms of epigenetic gene regulation?
DNA methylation
Histone modification
RNA-associated silencing
How are genes methylated?
Addition of methyl group to the C5 position of cytosine to form 5-methylcytosine (5MeC). Almost exclusively at CpG dinucleotides - two methylated cytosines diagonal to each other on opposing DNA strands.
What enzymes are involved in DNA methlation?
DNMT1 - maintains methylation of new strand during DNA replication
DNMT3A and DNMT3B - mainly embryonic and add initial methylation
What are short Non-coding RNAs ?
MicroRNAs small strands of RNA ~22 nucleotides long, interfere with gene expression at the level of translation. Bind to the 3′-untranslated regions of their target mRNAs, thereby inducing enzymatic degradation and preventing translation
What are long Non-coding RNAs ?
~200bp long and are thought to form ribonucleoprotein complexes that interact with chromatin, regulating histone modifications and the structural transformations that distinguish heterochromatin from euchromatin.
What is rRNA?
Component of cytoplasmic and mitochondrial ribosomes
What is snRNA?
9 in total and are components of spliceosome
What is tRNA?
cytoplasmic tRNA (encoded by nuclear DNA) and mitochondrial tRNA (mt-tRNA) (encoded by mitochondrial DNA) transfer the correct amino acid to the ribosome during translation
What is the core promoter?
most proximal pmromter of a gene - contains RNA polymerase binding site - the TATA box
What are Proximal promoter elements?
located ~250 bp upstream from TSS; contains many primary regulatory elements; site where general transcription factors bind
What is encoded by the mitochondrial genome?
37 genes coding:
two ribosomal rNAs (rRNAs)
22 transfer RNAs (tRNAs)
13 polypeptides subunits of the oxidative phosphorylation system
What are LCRs?
chromosome-specific low copy repeats of around 1-400Kb which are the substrate for NAHR
What are some “common” LCR/NAHR mediated deletions/duplications?
Williams-Beuren Syndrome - chromsome 7
Prader-Willi Syndrome/Angelman syndrome - chromosome 15
Charcot-Marie-Tooth (dup) / HNPP (del) - chromosome 17
Smith-Magenis (del) / Potocki-Lupski (dup) - chromosome 17
DiGeorge syndrome and reciprocal duplication - chromsome 22
What are the three mRNA surveillance mechanisms?
Nonsense Mediated mRNA decay pathway (NMD)
Nonstop Mediated mRNA decay pathways (NSD)
No-go Mediated mRNA decay pathway (NGD)
What therapies have been trialled to avoid NMD on PTC transcripts causing disease?
Increase PTC read through (happens 0.1-1% of time anyway)
Chimeric tRNAs, that specifically recognise the termination codon triplets and introduce an amino acid instead of termination
antisense oligonucleotides to remove PTC portion of a mutated transcript
NMD inhibitors
What are two example common pericentric inversion variants which class as normal variation?
inv(3)(p13p25) and inv(11)(q21q23)
What are the four common constitutive heterochromatic normal variants?
1qh, 9qh, 16qh and YqhWh
What are transposable elements?
units of DNA that move within the genome - either “copy and paste” or “cut and paste”
They account for 45% of genome. Most commonly Long Interspersed Nuclear Elements (LINES ~500,000 per genome) and Short Interspersed Nuclear Elements (SINES ~1,500,000 per genome).
How can transposable elements cause disease?
Insertion within a gene may disrupt a coding region and homologous recombination and/or mismatch repair between these elements may induce structural chromosomal rearrangements
What are some examples of Satellites?
Satellite DNA, Minisatellites and Microsatellites
What are fragile sites?
Segments of uncoiled chromatin that is liable to show gaps and breaks in chromosomes which are exposed by our efforts to control passage through the cell cycle and are normal variation
Give an example fragile site which is not classed as normal variation
Xq27.3 (FRAXA) and Xq28 (FRAXE) due to hypermethylation of FMR1
What sex chromosome aneuploidy is thought to always be mosaic? What types of mosaicism are observed?
Turner syndrome
Numerical - e.g. 45,X/46,XY or 45,X/46,XX/47,XXX
Structural - e.g. 45,X/46,X,i(X)(q10) or 45,X/46,X,+mar, 45,X/46,X,r(X)
What autosomal chromosome aneuploidy is always mosaic?
Pallister Killian syndromeW
What is Pallister Killian syndrome?
i(12p) - tetrasomy for 12p
Demonstrates tissue specific mosaicism with normal diploid complement in PB due to instability i(12)p at mitosis so is lost through rounds of mitosis. But significant levels in fibroblast, AF, CVS and BM
Buccal smears may be of use using interphase FISH or qPCR to detect the additional i(12)p
What is MMEJ ?
microhomology-mediated end joining
double stranded repair mechanism that results in deletions and insertions that flank the break sites -requires short regions of homology (5-25bp) at either side of a double strand break allowing repair to occur through annealing and ligation
What is the result of Breakage-fusion-bridge cycle ?
Dicentric chromosomes which are then pulled apart and break in anaphase. This will occur multiple times until a chromosome acquires a telomere
What is FoSTeS?
Fork stalling and template switching
3’ end of the lagging DNA strand disengage from the original template and anneal, by virtue of microhomology, to a single-stranded section of DNA in a nearby replication fork. Can result in deletions, inversion, duplication, translocations
What is Chromothripsis?
hundreds of genomic rearrangements occur in single one-off event to produce highly complex derivative chromosomes seen in some cancers
What is the most common reciprocal translocation?
t(11;22)(q23.3;q11.2)
Risk of der(22) offspring (Emmanuel syndrome) as result of 3:1 segregation
What are the two most common robertsonian translocations? And how do they arise?
rob(13;14) and rob(14;21)
centric fusion
How can UPD cause disease?
1) Genes within the UPD region are subject to genomic imprinting
2) Homozygosity for recessive mutations: two copies of a recessive mutation transmitted from a heterozygous carrier parent (‘isozygosity’)
3) UPD resulting from a somatic recombination can cause loss of heterozygosity (LOH) or loss of imprinting (LOI)
What are the different types of UPD?
When does each arise?
Uniparental isodisomy (UPID) - Meiosis II nondisjunction or mitotic error
Uniparental heterodisomy (UPHD) - Meiosis I nondisjunction
What occurs due to UPD for the entire chromosome complement?
Complete hydatidiform mole - paternal UPD (usually 46,XX)
Benign cystic ovarian teratoma - Maternal UPD
Partial hydatidiform mole - triploidy with extra set of either mat or pat chromosomes
How can complete chromosome UPD occur?
Trisomy rescue
Monosomic rescue
Mitotic error
What is trisomy rescue?
Meiotic nondisjunction in one parent results in a disomic gamete
Fertilisation of this disomic gamete with a normal haploid gamete results in a trisomic conceptus
Rescue’ through loss of one homologue (perhaps through anaphase lag) at a very early postzygotic stage (possibly even in the zygote), results in UPD in 1/3 cases (usually mat)
What is monosomic rescue?
Meiotic nondisjunction in one of the parents results in a nullisomic gamete.- Fertilisation of this nullisomic gamete with a normal haploid (monosomic) gamete results in a monosomic conceptus.
‘Rescue’ of the remaining homologue results in specifically isodisomy (usually pat)
What is mitotic error?
Conceptus and subsequent cell line are initially normal. Mitotic error leading to either trisomy or monosomy is then ‘rescued’ to result in isodisomy
What is the function of sodium bisulphite?
converts unmethylated cytosine nucleotides to uracil so can be used to differentiate between methylated and unmethylated DNA
What techniques for UPD detection use sodium bisulphite?
Methylation Sensitive (MS) PCR
Bisulphite restriction analysis and PCR
Methylation Sensitive (MS) melting curve analysis
Pyrosequencing
What techniques can detect UPD without sodium bisulphite?
Microsatellite Analysis
MS- MLPA
Southern Blotting with MS restriction enzymes
SNP array
WES/WGS
What is most common cause of 47,XXY offspring?
Paternal XY non-disjunction at MI accounts for 50% of XXY cases
What are the three major classes of eukaryotic pseudogenes?
Non-processed (duplicated) pseudogenes
Processed (retrotransposed) pseudogenes
Unitary (solitary) pseudogenes
Give an example a pseudogene can have at a DNA level
gene conversion/recombination with their parental homolog - e.g. between PSM2 and PMS2CL
Give an example a pseudogene can have at a RNA level
post-transcriptional regulation. e.g. PTENP1 (a pseudogene of PTEN) acts as a ‘miRNA decoy’ by binding to miRNA and allowing PTEN to escape miRNA-mediated silencing
What is the resolution of G banding?
3-5Mb
How does G banding work?
What underlies the pattern?
Treating aged/dried metaphase preparations with a protease (dilute trypsin) then staining with Giemsa stain
Heterochromatin stains darker whereas euchromatin stains light
What is R banding? What is main benefit?
Almost mirror of G - dark and light swap.
Allows visualisation of telomeres
What is Q banding?
Fuorescent technique using Quinacrine dihydrochloride which stains A-T rich sites
What is C banding?
C-banding stains constitutive heterochromatin
What is Cd staining?
pair of dots at each centromere, one on each chromatid
What is Replication banding?
How is it useful?
5’-Bromo-2’deoxyuridine (BrdU) is a thymidine analogue that is readily incorporated into chromosomes
Can be used to look for sister chromatid exchange that occurs during mitosis. Normally 5-8 SCEs per mitosis. In Bloom syndrome will be 10X more than this
What usually informs on whether adjacent-1 or adjacent-2 is likely to happen?
Adjacent-1 = the translocated segments are shorter than the centric ones
Adjacent-2 = the centric segments are shorter than the translocated ones
What are the two types of 2 break inversions?
Pericentric includes centromere
Paracentric does not include the centromere
What is the spliceosome?
Complex of snRNAs and their associated proteins
Outline the activity of the spliceosome
U1 snRNA binds to donor splice site
U2 snRNA binds to branch site (Adenine)
U1 and U2 come together to form a loop
U4, U5 & U6 bind and GT donor “attacks” branch point and AG is cleaved
Exons joined
How is splicing regulated?
trans-acting elements
cis-acting elements:
Exonic splicing silencer (ESS) - short region (usually 4-18 nucleotides) of an exon which recruit proteins that will negatively affect the core splicing machinery and involved in exon skipping
Exonic splicing enhancer (ESE) - DNA sequence motif consisting of 6 bases within an exon which enhance splicing
What are the three “box” promoters of transcription?
TATA box - 25-35bp from transcription start and indicates direction for transcription and strand to be read
GC box - ~110 bases upstream from the transcriptional start site
CAAT box - ~80bps upstream and strongest determination of promoter efficiency
What are enhancers?
cis-acting short sequence elements
bind gene regulatory proteins which loop DNA between promotor and enhancer to allow proteins bound to enhancer to interact with promoter bound TFs or RNA polymerase
What is 5’ capping?
Methylated nucleoside, 7-methylguanosine (m7G) is linked to the 5’ end of the RNA via a 5’-5’ phosphodiester bond
When does X inactivation happen?
Random process likely in late blastula stage and the inactivated X is inherited
How is X inactivated?
Xist is encoded by X inactivation centre (XIC) on Xq13 and codes for long non-coding (lnc) RNA molecule
Xist RNA molecule spreads along the chromatin outwards from the XIC. Resulting heterochromatin modifications such as histone deacetylation and methylation of CpG islands are epigenetic processes which organise the chromatin into a closed confirmation.
What regions escape X inactivation?
Genes which require bi-allelic expression in the pseudo-autosomal regions (coded for by both X in females and X and Y in males)
How can X inactivation effect other chromosomes?
X-autosome translocation. Expression of Xist on derivative chromosome will inactivate autosomal regions
What is skewed X-inactivation?
In theory should be totally random but can be skewed - either benefit (no phenotype from X-linked condition) or drawback (increased phenotype from X-linked condition)
What is most common method for X-Inactivation studies?
HUMARA Assay
PCR-based technique which amplifies a polymorphic CAG repeat region located within the first exon of the androgen receptor gene
Genomic DNA is first digested using HpaII, a methylation-specific enzyme that will only cut the unmethylated (i.e. active) allele therefore allowing us to distinguish between the active and inactive X chromosomes.
PCR primers are specific to regions flanking HpaII sites, such that digestion of the unmethylated allele disrupts PCR amplification. As a result, only the methylated (i.e. inactive) allele will give rise to a PCR product.
PCR products are then separated by capillary electrophoresis and amplification patterns between the undigested and digested alleles can be compared
