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FP_23_S1 > DNA_1 > Flashcards

DNA_1 Flashcards

1
Q

what is a DNA molecule?

A

Polymer consisting of 5 C sugar deoxyribose, phosphate group & nitrogenous base (heterocyclic ring of C & N)• Purines=A and G (2 interlocked rings) and Pyrimidines=C and T; a single ring

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2
Q

describe the structure of DNA

A

sugar-phosphate backbone linked by phosphodiester bonds (3’ P links to 5’P)N base links to 1’ C. base + sugar = NucleoSideNucleoSide + P = NucleoTideDouble helix bound by hydrogen bonds between complimentary bases A:T (2H bonds) & G:C (3H bonds)2 anti-parallel strands curve around each other resulting in major & minor groves1 turn = 3.6nm B (right-handed) DNA most abundant

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3
Q

describe the structure of RNA

A

single-strandedA pairs with Uracil more unstable due to Additional hydroxyl group at the 2’ position A-form helix

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4
Q

how is DNA packaged

A

Nucleosome = 2nm DNA coiled around 8 +charged histones 2 x (H2A + H2B +H3 + H4)Chromatosome = nucleosome + H1 (binds to linker DNA)Nucleosomes joined by linker DNAallows transcriptional activity chromatin (30nm fiber) = consists of nucleosomes packed into a spiral of 6-8 nucleosomes per turn Metaphase = DNA condensed to 1/10,000 (by topoisomerase II and condensins) Interphase chromatin is varied in compaction level

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5
Q

what are the 2 classes of heterochromatin?

A

 Constitutive: condensed and generally inactive. Consists largely of repetitive DNA Facultative: sometimes inactive (condensed) and sometimes active (decondensed) e.g. X-inactivation

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6
Q

what is the function on nc-RNA

A

help regulate expression of other genes

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7
Q

what is the Open Reading Frame?

A

sequence of nt triplets read as codons > amino acids. begin with initiation codon AUG methionine and ends with stop codon.

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8
Q

what are regulatory factors? give examples of diseases

A

required by RNA polymerase to initiate gene transcription. cis- acting (same DNA molecule as genes they regulate) and trans-acting (produced by remote genes and migrate to site of action). Some genes occur in clusters regulated by a locus control region.examples of diseases:LDLR promoter variant causes FHFMR1 5’UTR expansion causes gene methylation and promoter silencing in FRAX

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9
Q

Describe cis acting regulatory factors:

A

cis = same DNA molecule as genes they regulate. examples:Promoter: regulator region 5’ end of gene to which RNA polymerase binds to initiate transcription. consists of core promoter (most proximal) - contains RNA polymerase binding site, TATA box (30bp upstream of mRNA start site) where transcription factors and histones bind, and/or initiator element (specified transcription initiation to RNA polymerase) and transcription start site. defines transcription directionEnhancer: regulatory sequence that modulates rate of transcription in response to binding of activators. binding of regulatory proteins causes DNA between promoter and enhancer to loop out allowing interation of regulatory proteins with promoter TFs or RNA polymerasesilencer: repressors bind (inhibit activators) reducing transcription. prevent gene expression through cell-cycleinsulator: protects genes from inappropriate signals by blocking action of enhancer on promoter

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10
Q

Describe trans acting regulatory factors:

A

trans = produced by remote genes and migrate to site of action:transcription factors - controls rate of transcription by binding to specific DNA sequences

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11
Q

Describe the 5’ UTR

A

Regulates translation. spans transcription start site (TSS) to nt before mRNA start site & binds ribosome for polypeptide synthesis. 20% of genes express alternate 5’ UTRs by using multiple promoters to regulate gene expression. BRCA1 has 2 different transcripts derived from 2 different promoters which differ in 5’ UTRs. longer transcript predominantly expressed in breast cancers.

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12
Q

describe the 3’ UTR

A

Regulates translation. immediately follows translation stop codon and contains terminator sequence (endpoint for transcription and releases RNA polymerase) and regulatory regions (control polyadenylation, translation efficiency, stability and gene expression)

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13
Q

what is the polyadenylation signal

A

directs addition and cleavage of poly(A) tail to end of mRNA trancript - important for nuclear export, translation and mRNA stability. cleavage occurs 15-30 nts downstream from signal.

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14
Q

describe the mitochondrial genome?

A

transmitted exclusively through females. DS circular molecule containing 37 genes coding for 2 robosomal. 22 tRNA and 13 polypeptides (subunits of enzyme complexes of oxidative phosphorylation system). heavy and light chain transcribed from different promoter regions in opposite directions. genes closely clustered and contain no introns.

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15
Q

describe transcription?

A

5’ to 3’ synthesis of ssRNA by RNA polymerase II, complimentary to antisense DNA strand and same base sequence as sense strand (except T>U). Initiation: TFs (trans-acting) bind promoter (cis-acting)& position RNA polymerase II for RNA synthesis. RNA transcript undergoes splicing, capping and polyadenylation.

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16
Q

describe 5’ capping

A

occurs shortly after transcription initiation. a methylated nucleoside is added to 5’ end of RNA via phosphodiester bond. protects from exonuclease activity, facilitates transport to cytoplasm, facilitates RNA splicing and attaches to ribosome during translation

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17
Q

what is a ribosome?

A

RNA-protein complex composed of 60S subunit and 40S subunit

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18
Q

what is tRNA?

A

30 different types. up to 95nts, translates mRNA>protein. anticodon loop recognises complimentary mRNA codon. the amino acid is covalently linked to 3’ OH group by tRNA synthetase. only first two bases fit base-pairing rules, the last base is a “wobble” base as genetic code is degenerate.

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19
Q

describe the process of translation?

A
  1. initiation = 5’ cap of mRNA binds ribosomal small 40S subunit & scanned until start codon identified. initiator tRNA(met) pairs with AUG start codon and binds to P site of ribosome. binding of tRNA induces conformational change and transfer of peptide chain to A site occurs2. elongation: tRNA at A site moves to P site and used tRNA at P site then moves to E site and is released upon binding of next tRNA to to A site. 3. termination = elogation ends with stop codon. no complimentary tRNAs so hydrolysis of bond between tRNA and polypeptide at P side occurs and the polypeptide is released
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20
Q

give examples of errors in translation causing human disease

A
  1. BRCA1 longest 5’ UTR transcript expressed in cancerous tissue which is translated less efficiently so BRCA1 protein expression is inhibited in breast cancer tissue as opposed to normal tissue which contains both2. DM1 caused by triple expansion in 3’ UTR results in toxic GOF effect with translation dysregulation3. m.3243A>G tRNALeu(UUR) causes MELAS
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21
Q

what are ncRNAs?

A
  1. • constitute the majority of the human transcribed genome (60%)2. not translated into proteins3. • participate in complex networks of interactions with other nucleic acids and proteins 4. regulators of gene expression 5. • involved in many biologic processes: cancer, inflammation, and neurologic diseases
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22
Q

what are long nc RNAs?

A
  • > 200 nt long- > 20 000 have been identified- not evolutionarily conserved- similar to mRNA (often transcribed by RNA pol II, may be polyadenylated, can show complex splice patterns)- several types: antisense, intronic, exonic, promoter-associated- Biological processes: regulate gene expression in cis and trans, epigenetic, transcription, XI (XIST expressed in cis to silence x chromosome), genomic imprinting, RNA splicing
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23
Q

give examples of how long nc RNA cause human disease?

A
  1. H19 involved in imprinting at 11p15 Beckwith-Wiedermann syndrome2. UBE3A - ATS (antisense transcription) in angelmann3. SMA-AS1 recruits chromatin-modifying complexes in SMA
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24
Q

what is micro RNA?

A
  • small nc RNA 22nt long- highly conserved- regulate gene expression by post-transcriptional gene silencing- binds to 3’UTR of mRNA and either prevents translational machinery binding or promotes mRNA degradation through deadenylation of polyA tail- involved in proliferation, apoptosis, differentiation and development- resistant to RNases
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25
Q

what is si RNA?

A
  • small ds nc RNA ~20 nt long- downregulates expression of target genes- processed from long ds RNAs (through dicer)- can mediate gene silencing by direct heterochromatin formation
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26
Q

what is piwi interacting RNA (piRNA)?

A
  • small nc RNA ~30nt long- >23 000- controls gene expression- dicer-independent , expressed only in germline cells- 3 groups: transposon deived, mRNA derived and lnc-RNA derived- associate with PIWI proteins to create a silencing complex that induces degredation and methylation
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27
Q

what are circular RNA’s?

A
  • circular ss RNA molecules generated during back-splicing of mRNA to coavalently link 3’ end of an exon to 5’ end of upstream exon- resistant to RNase digestion so more stable than linear RNA molecules- regulate linear RNA transcription and protein production- dysregulation implicated in cancer occurence and progression
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28
Q

why might nc RNAs be useful as diagnostic tools?

A

NAME?

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29
Q

how can nc RNAs be used as therapeutic agents?

A

NAME?

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30
Q

what are antisense oligos?

A

ss nucleic acid sequences that target specific regions of pre-mRNA and modulate gene expression

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31
Q

give examples of antisense oligos used in gene therapy?

A
  • SMA: spinraza (Nusinersen) modifies SMN splicing by blocking intron 7 splice site to include exon 7 in SMN2 transcripts resulting in more full length SMN protein. injected into spinal cord as cannot cross blood-brain barrier- DMD - Exondys 51 - hybridises to exon 51 of DMD pre-MRNA causing it to be skipped during splicing which corrects the translational reading frame in certain DMD gene deletions resulting in shorter but functional protein- HD - HTTRx suppress translation of HTT mRNA containing CAG expansion - targets HTT snps so doesnt target expansions in other genesAngelmann - ASO against lncRNA UBE3A antisense transcript
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32
Q

what is a chromosome made of?

A

chromatin (DNA + protein)

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33
Q

what are the 3 types of chromosome structures?

A
  1. metacentric = q and p arms equal length2. submetacentric = arms unequal3. acrocentric = very short p armsautosomes numbered largest to smallest except chr21 smaller than 22
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34
Q

what is a centromere?

A

highly specialized chromatin provides foundation for kinetochore assembly (disc shaped protein structure) and site for sister chromatid attachment. sister chromatids remain attached until checkpoint is reached. attachment mediated by cohesin complex of proteins. as cell progresses to anaphase the cohesin is degraded allowing sister chromatids to be separated to opposite poles. most constricted region of mitotic chromosome

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35
Q

when does chromatid separation occur?

A

mitosis and meiosis II

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36
Q

describe diseases associated with centromere dysfunction?

A

1) premature centromere division (PCD) age dependent process occuring in women, leading to increase in x chromosome aneuploidy2) premature chromatid separation (PCS) - separate chromatids and discernible centromeres and involves most chromosomes in a metaphase. 40% of normal individuals. heterozygous PCS = >5% of cells and may cause decreased fertility and increase in aneuploidy in offspring. AD BUB1B mutation3) Roberts syndrome - chromosome breakage. LOF in ESCO2 (8p21.1) results in delayed cell division and incerased cell death. growth retardation, limb malformation, craniofacial, ID and cardiac abnormalities

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37
Q

what is the Kinetochore?

A

large multiprotein complex (>80) assembles on the centromere and acts as point of attachment for spindle. essential for segregation.

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38
Q

what is a neocentromere?

A

a new centromere that forms at an abnormal locationmay form on acentric preventing them being lost during cell division. formed via two processes1) inverted duplication of distal part of chromosome results in acentric marker2) interstitial deletion forms ring and linear marker chromosomeneocentromere then formed which lacks repetitive a satellite DNA and CENP-B. associated with cancer and MR

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39
Q

what is a telomere?

A

NAME?

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40
Q

what is the function of the telomere?

A

NAME?

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41
Q

what is the structure of telomeres?

A

-TTAGGG repeats(highly conserved) associated with telomere-binding proteins-adjacent to this are subtelomeric repeats (not conserved)- proximal to subtelomere repeats is chromosome-specific DNA and subtelomere- 3’ single stranded overhang 150-200 nt long can form telomeric loops to protect ends when replicating lagging strand

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42
Q

what is telomerase?what is its structure?what is its function?

A

NAME?

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43
Q

give an example of a disease associated with telomere malfunction?

A

cri du chat 5p deletopn = cat-like-cry, microcephaly, palmar creases. deleted region included hTERT genetelomerase reverse transcriptases which maintains telomere.

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44
Q

what is the Nucleolar organizing region (NOR)?

A
  • organizes nucleolus structure and contains 200 rRNA genes for protein synthesis- positioned on short arms of acrocentricscontains rRNA genes 5.8S 18S and 28S- if active it stains darkly with silver nitrate (Ag-NOR staining)
45
Q

what are replication origins?

A

NAME?

46
Q

what is a G-band evaluation score?

A

The minimum standard acceptable G band resolution for a given referral reason

47
Q

what causes the banding pattern in Giemsa staining? what are the differences between G-dark and G-light stains?

A

NAME?

48
Q

what is R banding?

A

NAME?

49
Q

what is Q banding?

A

NAME?

50
Q

what is C-Banding

A

(Constitutive heterochromatin banding).centromeric material comprised of repetitive DNA, satellite DNA (short tandemly repeated sequences: Alpha-satellite DNA, DNA satellite some non-repetitive DNA). It is differentiated from facultative heterochromatin in that facultative heterochromatin is condensed only semi-permanently via epigenetic changes which are reversible and can allow DNA transcription. Constitutive heterochromatin is permanently untranscribable.Constitutive heterochromatin is highly polymorphic, likely due to the instability of the satellite DNA. affects size and localisation of heterochromatin with no phenotypic effect. used to identify polymorphic variants in heterochromatic regions, inversions and rearrangements. useful to identify dicentric and pseudodicentric chromosomes and markers

51
Q

what is T banding?

A

telomeres

52
Q

what is a counter-stain?

A

Counterstaining is a technique that is used to induce banding with fluorochromes that bind and fluoresce uniformly throughout the chromosome. It is also used to enhance banding patterns that do not have a very high resolution. can stain 15p quick but fades quickly eg. DAPI

53
Q

what is replication banding? why is it useful for bloom syndrome? what are features of bloom syndrome?

A

BrdU is incorporated into chromosomes which is a thymidine analogue. Stained and then the BrdU DNA is stains different colour. used to detect early and late replications, detect different cycles of replication and count sister chromatid exchanges. used in Bloom syndrome where SCE no longer prevented after DNA damage due to BLM mutations leading to hyper-recombination and 10x more SCE. Bloom syndrome features = sunlight sensitivity, dwarfism, immunodeficiency, azoospermia and POF

54
Q

what is NOR staining?

A
  • stains p arms dark of acrocentrics with actively transcribed rRNA genes. stained with silver nitrate (ag-NOR). uses: see if marker has satellites, to check maternity/paternity as staining pattern is heritable). only technique for staining satellite stalks, cheaper than FISH. BUT siddifult to G band afterwards and very messy
55
Q

what is DNA replication? what are the three stages?

A

semi-conservative process where parental strands act as template for synthesis of new complementary strand. 3 phases:1. Initiation: begins at origins of replication, recognised by the origins recognition complex (ORC) in S phase. Topoisomerases nick DNA to be unwound by helicases. Allows RNA primer to bind followed by polymerase. Primers provide 3’ hydroxyl group for DNA polymerase to start synthesis. 2. Elongation - primers removed and replaced with nuclleotides and backbone sealed by DNA ligase. Two replication forks allows polymerase to move in opposite directions 5’ to 3’ adding dNTPs. Lagging strand is synthesized discontinuously - polymerase elongates a short stretch (okazaki) then moves to new primer as the helicase moves along. 3. Termination - RNA primers removed and gaps in okazaki fragments filled by polymerase D. Nicks are joined by DNA ligase completing fully replicated chromosome

56
Q

what Genetic Diseases are related to DNA replication?

A

Bloom syndrome - BLM gene. AR disorder caused by a helicase defect. symptoms = sensitivity to sunlight, growth deficiency, predisposition to malignancy & chromosomal instabilityPOLE in somatic cancer- produces polymerase without proofreading ability found in CRC and endometrial cancer

57
Q

what 3 conditions are required for DNA polymerase?

A
  1. 5’ to 3’ direction2. ss DNA only3. needs free 3’ end (provided by primase)
58
Q

what is an end replication problem?

A

no template at end of chromosome for primase to copy to make the RNA primer for the lagging strand.

59
Q

how is the end replication problem solved?

A

telomerase - reverse transcriptase has TERC subunit which is complimentary to telomere TTAGGG repeats. telomerase binds to 3’ lagging strand and acts as template to extend telomere. lagging strand now has room for primase and can be extended. lagging strand however cannot be extended to extreme 5’ end leaving an overhang which can form telomeric loop which protects telomere DNA from cellular mechanisms that repair ds-DNA breaks. In adult somatic cells, telomeric DNA does not get replicated and the telomeres shorten - aging.

60
Q

what are the phases of the cell cycle? G0, G1, S, G2 and M?

A

G0 = resting phaseG1, S, G2 = interphase:G1 = Growth phase where proteins and RNA made only. chromosome is a single double helix. At G1 checkpoint (restriction point) the cell is committed to division and moves to S phaseS= DNA synthesis replicates genetic material. each chromosome has 2 sister chromatids now. G2 = cell continues growing.G2 checkpoint - ensures enough cytoplasmic material for mitosis and cytokinesis (cell division)M = mitosis - cell stops growing. nuclear division and cytokinesis. Metaphase checkpoint M ensures cell is ready to complete division.

61
Q

what are the stages of mitosis?

A

prophase = nuclear membrane breakdown, chromosomes condense and spindle fibers appearMetaphase = align at centreanaphase - centromeres divide & sister chromatids move to opposite polestelophase = nuclear membranes formand chromosomes decondense + spindle disappearscytokinesis = cytoplasm divides and parent has become 2 daughter cells with identical genetic info

62
Q

how is the cell cycle controlled?

A

NAME?

63
Q

what are the steps for producing stained chromosome metaphase preparations FOR 72 hour synchronised culture in routine constitutional work ?

A
  1. mitogens induce cell division of resting cells eg. PHA2. synchronisation - inhibitors block cell cycle in S phase eg. thymidine3. block released (washed out) after 16-22 hours so cells continue through G2 together4. mitotic arrestants (colcemid) after 4.5 hours - stop cell division at metaphase and prevent spindle fibres forming5. ADD HYPOTONIC SOLUTION EG. KCl WHICH INCREASES VOLUME OF CELLS GIVING CHROMOSOMES MORE SPACE TO SPREAD. 6. Fix cells with fixative eg. Methanol:Acetic Acid (3:1) which kills cells and prepares them for bandingslide making - drop of cell suspension added to slide. As the fixative evaporates it enlarges cell and flattens out onto slide surface. Banding - slides aged in hot oven.Hanks solution - ageingtrypsin - enzyme digests chromosome and allows stainingLeishman’s stain - colours light and dark
64
Q

how does meiosis differ from mitosis?

A

two rounds of cell division to produce 4 daughter cells with half the number of chromosomes as original parent cell. daughter cells are not identical to parent cells unlike mitosis. MI = random independent assortment of chromosome pairs and crossover enables recombination. MII = separation of chromatids (to haploid state)

65
Q

what are the stages of meiosis?

A
  1. Prophase 1 - chromatin consenses, nuclear envelope breaks down, homologoues pair to form bivalents, crossing over between non-sister chromatids within homologue, homologues held by chiasmata2. metaphase 1 - bivalents align along metaphase plate and spindle forms3. Anaphase 1 - Homologous chromosomes drawn apart. Chromatids remain together4. Telophase 1 - haploid daughters (in females secondary oocyte has more cytoplasm than first polar body)5. Prophase/Metaphase II - nuclear envelope breaks down, new spindle and chromosomes (consisting of 2 chromatids align)6. Anaphase II - centromeres separate and sister chromatids migrate to opposite poles7. Telophase II - further cell division forms two haploid cells. in females you get a viable ovum and second polar body (non-viable)
66
Q

when does crossing over occur? what happens ?

A

meiosis prophase Itwo homologous chromosomes pair and equal exchange between two strands. sealed by DNA ligase

67
Q

what are chiasma?

A

connection where crossing over occurs

68
Q

what are potential negative consequences of recombination?

A

non-homologous crossover (high homology but are not alleles) - leads to loss or gain of materialsingle gene disorders caused by deletion or duplication of a single gene eg. BRCA1contiguous gene disorders - several genes deleted or duplicated alters dosagesegmental aneuploidy syndromes eg. Di George 22q11.2, PWS

69
Q

where does cell splicing occur?

A

nucleustranscription > premRNA> processed in nucleus by 5’ capping, poly Adenylation and splicing. splicing removess introns by endonucleolytic cleavage and ligation

70
Q

what is the spliceosome?

A

A 60S complex involving five snRNAs and their associated proteins

71
Q

how does the snRNA complex interact with pre-mRNA?

A
  1. U1 snRNA binds donor site2. U2 binds branch site and U1 and U2 join together to form lariat loop (transesterification reaction)3. U4, 5 and 6 associate with U1 and 2 to form spliceosome4. U5 binds both donor and acceptor sites and cleavage of acceptor site by transesterification joins exons together5. Lariat intron & spliceosome unbound
72
Q

what are the four classes of splicing regulatory elements?

A
  1. exonic splice enhancers - Generally hexamers and are evolutionarily conserved. Interact with SR (Ser-Arg) proteins 2. exonic splice silencers - Variable sequences that bind heterogeneous nuclear ribonuclear proteins (hnRNPs)3. intronic splice enhancers - Generally hexamers and are evolutionarily conserved. Interact with SR (Ser-Arg) proteins 4. intronic splice silencers - Variable sequences that bind heterogeneous nuclear ribonuclear proteins (hnRNPs)
73
Q

what is an An exonic splicing silencer

A

ESSs (cis-regulatory element) inhibit or silence splicing of the pre-mRNA and contribute to constitutive and alternate splicing (exon skipping). It does this by interfering with core splicing complex eg. U1 and U2

74
Q

what is an An exonic splicing enhancer ?

A

6 base DNA motif that enhances splicing - thought that they interact with U2 snRNA to do this. mutations in this sequence cause genetic disorders and some cancers

75
Q

what is alternative splicing?

A

mechanism to regulate gene expression in eukaryotes. contributes to proteomic diversity because it allows for the generation of multiple proteins from a single gene. Most common is exon skipping. also alternative 5’ or 3’ splice sites, intron retention and mutually exclusive exons (different combinations of exons), alternative promoters and alternative polyA leading to proteins with different functions or activities

76
Q

how can splicing defects impact on disease?

A

up to 50% of human disease mutations alter splice elements with 10% due to consensus splice mutations.1. disruption of splicing elements - non-coding SNVs in BRCA, Ataxia telangiectasia, Retinitis pigmentosa. consensus change may cause exon skipping or intron inclusion. Branch site mutation = Ehlers-Danlos type II. disruption of cis-elements ESS/ESE eg. 3bp in-frame deletion in exon 3 of MLH1 causes disruption of an ESE and causes HNPCC. 1/4 of SNVs in exons 9 & 12 of CFTR result in abnormal splicing 2. toxic RNA eg. unstable repeat expansions cause dysregulation of alternative splicing such as DM 3’ UTR CTG GOF or In DM2, CCUG GOF expansion affects the non-coding regions of the ZNF9 gene.3. Mutations (trans-acting) affecting splicing factors - eg. ALS, DCM - inclusion of differentially expressed exons. CFTR intron 8 poly T and TG tracts cause exon-skipping. SMA - exon 7 SNV promotes exon skipping and production of a truncated, inactive protein.

77
Q

what therapies are available to target splicing defects?

A
  1. antisense oligos can be used to enhance or repress eg. blocking splice sites for exon skipping in DMD or ptromote exon 7 inclusion in SMN2. can also be used to target mutant isoform trancripts for degradation eg. CTG GOF repeat in DM1
78
Q

what is no-Go decay?

A

mRNA transcripts on which ribosomes have stalled (e.g. due to secondary structures)

79
Q

where in the gene escapes NMD?

A

last and 3’ 50bp of penultimate exon and within first 100nt (uses alternate start codon)

80
Q

describe mechanism for NMD?

A

exon junction complex proteins are normally stripped off mRNAs by translating ribosome. If a PTC is present it remains bound. once ribosome reaches PTC, the SURF complex is formed which interacts with EJC and NMD regulators to form mRNA decay-inducing complex (DECID) leading to mRNA degradation. OR premature release of ribosomes tags downstream mRNA for destruction

81
Q

give examples of diseases caused by NMD?

A

DMD - out of frame mutation leads to mRNA degradation. BRCA1 X mutations inactivate tumour suppressionALS caused by FUS mutations leads to neuron death

82
Q

what treatment is available forPTC? what issues are there?

A

read-through eg. Translana for DMD caused by nonsense variantssplice-switching oligos cause exon skipping eg. restore correct reading frame for DMDNMD inhibitors - eg. increased expression of TP53o Issues include delivery, toxicity, variability in cells, tissues, individuals

83
Q

what is non stop-mediated decay?

A

Detection and decay of mRNA transcripts which lack a stop codon. May be due to premature 3’ adenylation where ribosomes translate into 3’ region and stall and cannot eject the mRNA. Nonstop mediated decay mediates this problem by both freeing the stalled ribosomes and marking the nonstop mRNA for degradation in the cell by nucleases

84
Q

how are premature stop codons distinguished from natural ones?

A

by exon junction complexes - if there are EJCs present downstream of mutant PTC this will trigger NMD

85
Q

how does read-through gene therapy work?

A

tRNA with 2/3 complementary bases anneals to incorrect stop codon and is incorporated generating the full length peptide

86
Q

what is ribosomal RNA? what disease is rRNA involved in?

A

a type of non-coding RNA which is the primary component of ribosomes (cytoplasmic and mitochondrial). it is bound to ribosomal proteins to form small and large ribosome subunits and is involved in translating mRNA into protein (via tRNA). Involved in Treacher Collins syndrome - mutations involved in ribogenesis

87
Q

what are Small nuclear RNA and what disease are they involved in?

A

part of spliceosome complex, specifies where splicing occurs and involved in SMA-

88
Q

what are Small nucleolar RNAs (snoRNAs) and what diseases are they involved in?

A

guide chemical modifications and processing of other RNAs. involved in Prader-willi syndrome - paternal loss of snoRNAs in SNRPN locus responsible for phenotypic features of PWS

89
Q

what are tRNA’s and what disorders are they involved in?

A

transfer the correct amino acid to the ribosome during protein synthesis. cytoplasmic and mitochondrial. Cytoplasmic has cloverleaf structure with 3 hairpin loops including the anticodon loop. Involved in the diseases MELAS (Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) caused by MTTL1 SNVs and MERRF (myoclonic epilepsy with ragged red fibers) m.8344A>G in mt-tRNALys gene.

90
Q

what is tRNA wobble base pairing?

A

3rd base in anticodon can form H bond with several bases at 3’ position of the codon

91
Q

what is a heteromorphism?

A

normal variation of visible variant differing in size, morphology or staining properties (5% of human genome is structurally variable)

92
Q

what types of normal variation is there in the human genome?

A
  1. heteromorphism2. recurrent balanced translocations3. inversions - several pericentric classed as variants4. supernumerary marker chromosome - many show karyotypic variation without phenotypic effect5. Constitutive heterochromatic variation - AT rich, varies in length, satellitte DNA6. acrocentric p arm7. CNVs (>1kb) - dependent on gene content, breakpoints and insertion sites and regulatory elements. If >1%, inherited from normal parent more likely benign BUT may have variable penetrance and expressivity. 8. euchromatic variants - resemble duplications, but polymorphic. 9. repeat sequence polys10. transposable elements - units of DNA that move within genome eg. LINES and SINEs (such as Alu element). may be associated with disease if disrupt coding region11. satellite DNA/Variable Number Tandem Repeats (VNTRs)12. mini and microsatellites13. low copy repeats/segmental duplications - not associated with disease but NAHR may be pathogenic14. epigenetic variation - methylation and acetylation can alter DNA structure and expression15. fragile sites - uncoiled chromatin, low in gene content. can be silenced by hypermethylation16. polymorphism eg. SNV. not stable over time17. RFLPs - cleavage of DNA with restriction enzymes18. ExAC/GnoMad, dbSNP. DGV, Decipher
93
Q

by what 3 methods do mutations arise?

A
  1. DNA damage2. Recombination/replication errors3. failure to repair eg. checkpoint errors
94
Q

what is DNA damage ?

A

NAME?

95
Q

what is DNA replication error?

A

DNA polymerase adds incorrect NT during replication and some mutations evade the 3’→5’ exonuclease “proofreading” enzyme. uncorrected errors range from 1 x 10-4 to 1 x 10-6 mutations/gamete for a given gene.

96
Q

what DNA repair mechanisms exist?

A
  1. direct reversal - enzymes reverse the chemical reaction eg. removal of methyl group2. Excision repair - non defective strand used as template and damaged DNA is removed and replaced. Base excision repair, nucleotide excision repair and MMR3. ds-break repair - joining free DNA ends (however may place oncogene next to promoter). Homologous recombination repair (HRR) and non-homologous end joining (NHEJ)
97
Q

what is base excision repair?

A

NAME?

98
Q

what is Nucleotide excision repair (NER)?what syndromes are associated with this system?

A

major cellular defence system against the carcinogenic effects of UV exposure that Removes pyrimidine dimers caused by UV radiation. 1. detects damage2. nuclease excision of erroneous DNA section3. fills gap with DNA polymerase3. seals nick Xeroderma pigmentosum (XP), Cockayne syndrome (CS)

99
Q

what is mismatch repair?

A

NAME?

100
Q

what is Homologous Recombination Repair (HRR)? what diseases are associated?

A

NAME?

101
Q

what is Non-homologous Repair (NHR)?

A

Repairs DNA breaks without homologous templateNon-replicative mechanisms:1) non-homologous end joining - ligation of two DSBs2) brakage-fusion bridge cycle - sister chromatids that lack telomeres fuse to create dicentric chromosome. During anaphase they are pulled apart and cycle repeatsReplicative mechanisms:1) replication slippage - trinucleotide or dinucleotide expansion during replication2)fork stalling and template switching3) c) Microhomology-mediated break induced replication (MMBIR)

102
Q

what is a pseudogene?

A

A DNA sequence that resembles a gene but has been mutated into an inactive form over the course of evolution. It often lacks introns and other essential DNA sequences necessary for function.Group of long non-coding RNAs (lncRNA). They can positively or negatively regulate gene expression and are often aberrantly expressed in cancer

103
Q

what are the 3 types of pseudogene?

A
  1. processed (72% of pseudogenes) = derived from reverse transcription of mRNA, inactivate coding ability2. unprocessed - segmental dups and defective3. accumulation of mutations so loses coding capacity
104
Q

Example of pseudogene as positive gene regulator on cancer?

A

PTENP1 - functions as tumour suppressor and upregulation causes growth inhibition of tumour cells. regulates parent gene PTEN. Implicated in breast cancer, melanoma

105
Q

describe the SMN2 pseudogene

A
  • non-processed centromeric pseudogene. high sequence homology to SMN1 - distinguished by single SNVs in exons 7 and 8 and 3 intronic. all are synonymous but the exon 7 SNV disrupts ESE leading to aberrant splicing of SMN2 exon7. SMN2 retains partial functionality. number of copies acts as disease modifier. increased SMN2 = milder and increased life expectancy. SMN1 can convert to SMN2.
106
Q

what is the Chiasmata

A

Join bivalents together at locations along the length of the chromatids.

107
Q

what is Cohesin

A

Joins sister chromatids together, and also helps to maintain chiasmata.

108
Q

how can aneuploidy arise?

A
  1. meiosis i) 1:1:1 or meiosis ii) 2:12. mitosis - somatic or acquired results in mosaicism. less frequent than meiotic and mostly due to anaphase lagging.

FP_23_S1 (7 decks)

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