Basic Molecular Biology Flashcards
(180 cards)
1
Q
What is the structure of DNA?
A
Deoxyribose Sugar: A 5-carbon sugar called deoxyribose.
Phosphate Groups: One to three phosphate groups attached to the deoxyribose.
Nitrogenous Bases: Four possible bases—adenine (A), guanine (G), cytosine ©, or thymine (T).
2
Q
What is the structure of RNA?
A
RNA, ribonucleic acid has an additional hydroxyl group at the 2’ carbon which makes it more unstable.
Adenine pairs with Uracil instead of thymine
Single stranded
3
Q
How does DNA form a double helix?
A
Two DNA strands are held together by hydrogen bonds to form a duplex.
Bonds occur between complementary base pairs. A with T (2 hydrogen bonds), G pairs with C (3 hydrogen bonds).
The two strands run anti-parallel to each other and curve around each other to produce a minor and major groove. A single complete turn of helix (pitch) is 3.4nm, the distance between two base pairs is 0.34nm
4
Q
What are the different conformations of DNA?
A
B-DNA
A-DNA
Z-DNA
Cruciform and Hairpin Structures
H-DNA
G4-DNA
5
Q
What is B-DNA?
A
Right-handed helix (spirals in clockwise direction away from observer) and has 10 base pairs per turn. Most abundant type of DNA, commonly known as Watson and Crick model. Bacterial and eukaryotic cells adopt this form.
6
Q
What confirmation does RNA have?
A
A-DNA: right-handed helix with 11 base pairs per turn. RNA adopts A form. Not found in vivo (DNA)
7
Q
What are histones?
A
DNA wraps around histones which are rich in lysine and arginine and have strong positive charge- affinity for negatively charged DNA
Core histones: H2A, H2B, H3, H4
Linker histone H1
8
Q
What is a nucleosome?
A
The fundamental unit of DNA packaging
147bp of 2nm DNA helix coiled in less than two turns around a central core of 8 histone (2 each of H2A, H2B, H3 & H4) forms 10nm thick nucleosome filament
Adjacent nucleosomes are connected by short lengths (8-114bp) of linker DNA – length varies between species & different regions of genome
9
Q
What are chromatin?
A
Consists of nucleosomes packed into a spiral arrangement with 6-8 nucleosomes/ turn.
H1 histones are bound to the inside of the spiral with one H1 molecule associated with each nucleosome.
10
Q
What are euchromatin?
A
Euchromatin: relatively extended conformation “open” DNA under active transcription. Marked by weak binding of H1 histones and acetylation of the histones.
11
Q
What are heterochromatin?
A
Highly condensed throughout cell cycle, “closed” DNA and genes not expressed- associated with tight H1 histone binding
Can be constitutive: mainly repetitive regions which remains condensed and inactive
Can be facultative: at regions requiring dynamic regulation
12
Q
What is CTCF?
A
CTCF domains, bound by CTCF, pair to each other and bind with rings of cohesin to form chromatin loops.
Looping is powerful mechanism of gene regulation as loops can bring together distantly located regulatory sequences and their targets.
Genes within loops are expressed at higher levels than those outside loops.
Aberrant looping has been associated with disease.
13
Q
What are Topologically associated domains (TADs) ?
A
Loops interact with each other to form sub-topologically associated domains (sub TADs) which then interact with each other to form TADs, these form compartments and chromosome territories.
Organisation into TADs facilitates physical contacts between genes and their regulatory elements => important role in regulation of gene expression
Deletion of TAD boundaries may result in long range contact and misregulated expression => CTCF disruption changes TAD structure
14
Q
What is histone modification?
A
N-terminal tails of core histones protrude from nucleosomes
Specific amino-acids in the tails (and less often other regions of histone) can undergo various types of post-translational modification e.g. acetylation, phosphorylation and methylation etc.
Binding of different proteins to the chromatin affects condensation and local level of transcriptional activity
15
Q
What is histone acetylation?
A
Occurs on lysines; almost always associated with activation of transcription (e.g. acetylated H3K9 is found in actively transcribed promoters)
Addition of an acetyl group to lysine neutralises its positive charge and weakens interactions between histones and DNA, de-stablising chromatin architecture
16
Q
What regulates histone acetylation?
A
Regulated by the opposing action of histone acetyl-transferases (HATs) and histone deacetylases (HDACs)
17
Q
What is histone phosphorylation?
A
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
18
Q
What regulates histone phosphorylation?
A
Controlled by kinases (adds phosphates) and phosphatases (removes phosphates).
19
Q
What is histone methylation?
A
Takes place commonly on lysines and sometimes on arginines
Exists in 3 states: mono-, di-, tri- methylated.
Effect (transcription, repression or activation) depends on the amino acid residue modified and the number of methyl groups added:
- Lysine methylation can be involved in both repression (e.g. H3K9 & H3K27) and activation (e.g. H3K4).
- Arginine methylation has been implicated in transcription repression (e.g.H3R8) and activation (e.g. H4R3)
20
Q
What regulates histone methylation?
A
Facilitated by histone methyltransferases (HMTs), which recruit S-adenosylmethionine as a co-substrate for transfer of the methyl group
One possible arginine demethylase exists. Many lysine demethylases have been described.
21
Q
What other histone modifications are there?
A
Ubiquitination: Ubiquitin (a 76 aa polypeptide) is attached to histone lysines. Roles in transcriptional repression, activation and response to DNA damage
Sumoylation: attachment of small ubiquitin-like modifier molecules to histone lysines. Associated with repressive functions.
Deamination: converts arginine to a citrulline. Neutralises positive charge of arginine.
ADP-ribosylation
Proline isomerisation
22
Q
What are chromatin remodellers?
A
Multiprotein complexes that modify association of DNA and histones
Provide access to underlying DNA to enable transcription, chromatin assembly, DNA repair, DNA replication, DNA recombination, chromosome segregation and dosage compensation.
23
Q
What are the 5 families of chromatin remodellers?
A
SWI/SNF
ISWI
NuRD/ Mi-2 CHD
INO80
SWR1
24
Q
What is the role of DNA methylation in cancer development by influencing gene expression?
A
Both hypermethylation (excessive methylation) and hypomethylation (reduced methylation) are seen in tumour development.
DNMT (DNA methyltransferase) overexpression leads to hypermethylation of CpG islands at gene promoters. This can silence tumour suppressor genes
Seen in lung, colorectal cancer, gastric cancer
25
What are chromosomes?
Chromosomes consist of chromatin fibre (DNA and protein complex) folded and coiled into a compact arrangement in the nucleus of cells.
Chromosomes are constricted at a point called the centromere. This divides the chromosome into two arms, p arm (short) and q arm (long). Chromosomes are capped by telomeres.
26
What are the four types of chromosomes?
Metacentric
Submetacentric
Acrocentric
Telocentric (only centromere and long arms- not in humans)
27
What is the centromere?
The centromere is a region of highly specialized chromatin that has two key functions
1) assemble the kinetochore
2) maintain sister chromatids together before chromosome separation. It is easily visualized as the most constricted region of a condensed mitotic chromosome.
28
What is the function of the centromere?
The two key functions of the centromere are to assemble the kinetochore and maintain sister chromatids together before chromosome separation.
29
What is the structure of a centromere?
Composed of six classes of repetitive DNA: α-satellite (most abudant), β-satellite, ɣ-satellite, and three shorter motifs termed HSATI, HSATII, and HSATIII.
30
What is a higher-order array?
Most α-satellite DNA are organized into higher-order arrays (HOR) consisting of discrete units of monomers repeated in tandem.
While most human chromosomes harbor more than one related α-satellite HOR array, only one HOR array is typically associated with the kinetochore, and these are defined as “active” α-satellite HOR arrays.
31
What diseases are associated with centromere dysfunction?
- Premature centromere division (PCD) – age-dependent phenomenon occurring in women, characterised by rod-shaped X chromosome(s) without discernible centromeres,
- Premature chromatid separation (PCS) – consists of separate and splayed chromatids with discernible centromeres and involves all or most chromosomes of a metaphase. When PCS is present in 5% or more of cells, it is known as the 'heterozygous PCS trait' and has no obvious phenotypic effect, although some have reported decreased fertility and possible increase of aneuploidy in offspring.
32
What is the Kinetechore?
The kinetochore is a large multiprotein (>80) complex with a plate-like structure that assembles on a centromere and acts as a point of attachment for the microtubules/spindle fibres. It is essential for proper chromosomal segregation during mitosis or meiosis.
A kinetochore is positioned on the side each sister chromatid, facing the spindle pole to which the chromosome will be drawn during anaphase. Multiple microtubules appear to insert into the kinetochore.
33
What is the structure of the kinetochore?
- an inner kinetochore, which is tightly associated with the centromere DNA and assembled in a specialized form of chromatin that persists throughout the cell cycle;
- an outer kinetochore, which interacts with microtubules; the outer kinetochore is a very dynamic structure with many identical components, which are assembled and functional only during cell division.
34
What is a neocentromere?
A neocentromere is a new centromere that forms on a chromosome at a location that is normally not centromeric — usually as a result of disruption of the natural centromere.
- Can spontaneously form on acentric chromosome fragment preventing them being lost during cell division.
- Whereas most natural centromeres contain highly repetitive sequences, neocentromeres usually possess unique sequences-
35
What are telomeres?
Telomeres are highly conserved gene-poor, tandem nucleotide repeats in complex with telomere associated proteins (nucleosomes, shelterin complex, and chromosomal transcription factors) cap the ends of eukaryotic chromosomes and are required to protect chromosomal ends.
36
What is the function of telomeres?
- Maintain structural integrity – if lost the chromosome end is unstable. it can fuse with other broken chromosomes, be involved in recombination or be degraded.
- Prevents shortening of the chromosomes at each round of cell division- would result in cell death.
- Length of the telomere can also provide a counting mechanism that drives replicative senescence by limiting the mitotic potential of cells.
- Are important for chromosome positioning as they help to establish the 3-D architecture of the nucleus and aid chromosome pairing.
37
What is the structure of a telomere?
-consists of 3kb to 20kb of tandem TTAGGG repeats
- Located immediately adjacent to the TTAGGG repeats are the telomere associated repeats (TAR), also known as the sub-telomeric repeats, which are 100-300kb in size.
- Proximal to the TAR lies unique chromosome-specific DNA, commonly referred to as the sub-telomere.
- the telomere has a single-stranded overhand at its 3’ end around 150-200 nucleotides long due to the lagging strand being difficult to replicate.
38
Why is there a 3' overhang on telomeres?
Replication of linear DNA presents a problem in that DNA synthesis works in the 5' to 3' direction; this is ok for the leading strand but is opposite to the direction of the lagging strand. A succession of ‘back-stitching’ syntheses is required to produce a series of DNA fragments (Okazaki fragments) whose ends are then sealed by DNA ligase to ensure continuity of synthesis along the lagging strand.
39
What diseases are associated with telomere malfunction?
- Dyskeratosis congenital (DC): Rare inherited disorder with increased incidence of cancer. Characterized by abnormal skin pigmentation, nail dystrophy,
- Cri du Chat syndrome (CdCS) - Deletion of 5p (including TERT) Characteristic phenotype including cat-like cry, microcephaly, distinct facies and palmar creases.
- Anaplastic anaemia- Characterized by hypocellular bone marrow and low blood cell counts.
40
What is a Nucleolar organizing region (NOR)?
The nucleolar organising regions (NOR) are responsible for organising the nucleolus structure and contain the approx. 200 rRNA genes necessary for protein synthesis.
- Human NORs are positioned on the short arms of the acrocentric chromosomes.
- It contains ribosomal RNA (rRNA) genes 5.8S, 18S and 28S, which are organised on a 13kb transcription unit.
41
What are replication origins?
- Cis-acting DNA sequences which bind proteins in preparation for DNA replication.
Recognised by a six protein complex = ORC (Origin of Replication Complex) ORC1-6
42
What is G-banding and why is it widely used in clinical settings?
G-banding is a method for staining chromosomes that is simple, reliable, and reproducible. It is widely used because it requires only a light microscope for visualization and provides a standard framework for describing gene locations and chromosomal features.
43
Describe the staining process involved in G-banding
G-banding involves treating aged metaphase chromosome preparations with a protease (dilute trypsin) or hot 2x SSC, followed by staining with Giemsa stain or similar chromatin stain (e.g., Leishman or Wright stain).
44
What is the difference between heterochromatin and euchromatin based on G-banding pattern?
Heterochromatin appears as darkly stained bands and is composed of condensed, tightly coiled chromatin. It is genetically inactive, mostly constituted of repetitive DNA, and late-replicating.
Euchromatin appears as lightly stained bands and is composed of open, loosely packed chromatin. It is gene-rich, actively expressed, and early-replicating.
45
Explain the concept of the haploid set in G-banding evaluation
The haploid set is the summed total of resolvable bands from one homologue of each chromosome. It defines the lowest standard acceptable G band resolution for a given referral reason in chromosome analysis according to best practice guidelines.
46
What is the maximum resolution of G banding?
The maximum resolution of G-banding is 3~5Mb, meaning abnormalities smaller than this will be indistinguishable.
Often 10Mb in cancer chromosomes
47
What is R-banding and how does it differ from G-banding?
R-banding is an alternative to G-banding that produces a complementary banding pattern. Dark G-bands appear light with R-banding. It is less frequently practiced but offers improved visualization of telomeric regions if involved in aberrations.
48
What is the protocol for R banding?
The generalised protocol for R-banding involves incubating chromosomes in a hot (85-90°C) phosphate buffer followed by Giemsa or acridine orange staining.
49
Describe the staining protocol for C-banding
C-banding involves treating metaphase preparations with an alkali solution (usually Barium hydroxide) prior to staining to reveal constitutive heterochromatin.
50
What are the uses of C banding?
Constitutive heterochromatin is highly polymorphic, likely due to the instability of the satellite DNA.
Used to identify/confirm polymorphic variants in the lengths of the heterochromatic regions (1qh,9qh,16qh,Yqh)
It is also useful for identifying dicentric and pseudodicentric chromosomes and for studying marker chromosomes.
51
What is the purpose of Cd staining, and how does it differ from C-banding?
Cd staining produces a pair of dots at each centromere, specific to the centromeric region, allowing differentiation of functional from non-functional centromeres and to study Robertsonian translocations (centromere to centromere translocations of acrocentric chromosomes. It differs from C-banding in that it only stains active or functional centromeres
52
Explain the significance of nucleolar organizer region (NOR) staining and its method
NOR staining identifies chromosomal regions crucial for nucleolus formation. It involves staining with silver nitrate, with active transcription regions staining darkly. It's used to investigate rearrangements/polymorphisms of acrocentric chromosomes and differentiate fragile sites from inserted NORs.
53
What is the mechanism behind Replication banding/Sister Chromatin Exchange (SCE) staining?
Replication banding involves incorporating BrdU into synthesizing chromosomes, visualized by quenching fluorescence with Hoechst 33258 or by staining with Giemsa. Sister Chromatin Exchange (SCE) staining detects exchanges of genetic material between sister chromatids, indicating breakage syndrome when increased.
54
How is Replication banding/SCE staining used in studying sex chromosome abnormalities?
It is used to label early or late-replicating DNA, helping study sex chromosome abnormalities, such as the inactive-X being later replicating.
55
What is the definition of a gene, and how are genes classified?
A gene is a region of DNA used as a template to synthesize a functional complementary RNA molecule. Genes are classified into coding and non-coding categories.
56
Explain the difference between coding and non-coding genes
Coding genes are transcribed and translated to generate corresponding polypeptide sequences (mRNA), while non-coding genes do not serve as templates for making polypeptides but instead help regulate the expression of other genes.
57
What are pseudogenes, and what distinguishes them from functional genes?
Pseudogenes are non-functional genetic elements similar to functional genes but are unable to produce functional proteins due to genetic alterations, such as frameshift or nonsense variants.
- ~10,000 in mammalian genome (~20% are transcribed into RNAs)
58
Define Open Reading Frame (ORF) and its significance
An Open Reading Frame (ORF) is a sequence of successive nucleotide triplets read as codons specifying amino acids. It begins with an initiation (start) codon (AUG) and ends with a stop codon (UAA, UAG, or UGA), crucial for identifying coding regions in genes.
59
Describe the types of regulatory factors involved in gene transcription
Cis-acting (located on the same DNA molecule) and trans-acting (produced by remote genes)
Cis-acting factors include promoters, enhancers, silencers, and insulators, while trans-acting factors include transcription factors.
60
What is the function of a promoter, and what are its key components?
A promoter is a regulatory region close to the 5' end of a gene where RNA polymerase binds to initiate transcription. It consists of core promoter elements (including the TATA box and transcription start site), proximal promoter elements (250bp from start site where transcription factors bind), and distal promoter elements (further upstream, regulatory).
61
Explain the role of enhancers, silencers and insulators in gene regulation
Enhancers modulate the rate of transcription by binding specific proteins (activators), while silencers inhibit activators, reducing transcription. They play crucial roles in regulating gene expression during differentiation and the cell cycle
62
Differentiate between cis-acting and trans-acting regulatory factors
Cis-acting factors are located on the same DNA molecule as the genes they regulate, while trans-acting factors are produced by remote genes and migrate to the site of action. Transcription factors are examples of trans-acting regulatory factors.
63
What is post-transcriptional processing, and what is one example of it?
Post-transcriptional processing refers to the series of processing reactions undergone by RNA transcripts to form mature mRNA or non-coding RNA. One example is RNA splicing, which removes intronic RNA segments and joins exonic RNA segments.
64
Describe the structure and function of 5' and 3' UTRs in mRNA molecules
The 5' UTR spans from the transcription start site to the nucleotide before the mRNA start site and binds ribosomes. The 3' UTR immediately follows the stop codon and contains regulatory regions influencing mRNA polyadenylation, translation efficiency, localization, and stability
65
What is an insulator?
Sequence element that protects genes from inappropriate signals emanating from their surrounding environment either by blocking the action of enhancer on the promoter (if situated between them) or by acting as “barriers” that prevent the advance of nearby condensed chromatin
66
What initiates DNA replication, and where does it begin?
DNA replication begins at specific locations called origins of replication (OR) with the formation of the replisome.
67
Describe the role of the Origin Recognition Complex (ORC) in DNA replication initiation
The ORC, composed of six subunits encoded by genes OCR1-ORC6, binds to the origin of replication during the G1 phase of the cell cycle. It recruits and loads the MCM2-7 helicase onto DNA, forming a pre-replication complex.
68
What is the Domino model of replication, and how does it influence replication timing?
The Domino model suggests that replication begins at sites with an 'open' chromatin configuration, primarily in actively transcribed euchromatin, and later progresses to heterochromatic regions. Different pre-replication complexes are activated and initiate replication at different times during S-phase.
69
Explain the process of replication initiation complex formation
Addition of CDC45 and the GINS complex to the pre-replication complex forms the pre-initiation complex. This process, requiring cyclin-dependent kinase activity, recruits DNA polymerases α and δ, initiating replication and assembling the replisome at the replication fork
70
What is the role of topoisomerases in DNA replication?
Topoisomerases create nicks in a single DNA strand, relieving tension and allowing the double helix to be unwound by helicases, forming a Y-shaped replication fork
71
How are primers synthesized during DNA replication, and why are they necessary?
Primases attach small complementary RNA sequences as primers at the replication fork. Primers provide a 3' hydroxyl group needed by DNA polymerase to start synthesis.
72
Describe the function of DNA polymerases in DNA replication
DNA polymerases synthesize new DNA strands by adding deoxynucleoside monophosphate (dNMP) to the free 3' hydroxyl group of a growing DNA strand, using deoxynucleoside triphosphates (dNTPs) as substrates.
73
What are the main rules limiting the function of DNA polymerase?
DNA polymerase synthesizes DNA in the 5' to 3' direction, acts on single-stranded DNA provided by helicase, and requires a free 3' end provided by primase
74
What are the types DNA polymerases?
- DNA polymerase α (alpha) – complex of polymerase and primase that initiates DNA synthesis and Okazaki fragments
- DNA polymerase δ (delta)– main polymerase that synthesises most of the lagging strand, and is involved in DNA repair
- DNA polymerase ε (epsilon) – main polymerase that synthesises most of the leading strand, and is involved in DNA repair
In higher eukaryotes both δ and ε consist of heterotetramers (catalytic subunit, POLD1 and POLD2) and accessory subunits (POLD2/3/4 and POLE 2/3/4).
- Polymerases δ and ε also play essential roles in repair of chromosomal DNA in several repair pathways (NER -Nucleotide Excision Repair, BER - long patch Base Excision Repair, MMR – Mismatch Repair, DSBR - Double-Strand Breaks Repair, and BIR - Break-Induced Recombination).
75
Explain the significance of DNA polymerase families A, B, X, and Y
Different DNA polymerase families have distinct functions and error rates.
- Family A includes DNA polymerase γ (gamma) which replicates mitochondrial DNA
- Family B polymerases replicate nuclear DNA e.g. α, δ, and ε
- X and Y have very high error rates, wprk in DNA repair process so only synthesise small stretches. Also synthesise through stalled replication forks- translesion synthesis
76
What are the three stages of DNA replication?
Initation
Elongation
Termination
77
Explain the process of DNA replication elongation
During DNA replication elongation, the leading strand is synthesized continuously in the same direction as the movement of the replication fork, while the lagging strand is synthesized discontinuously in the opposite direction. Small DNA segments called Okazaki fragments are formed on the lagging strand and later joined by DNA ligase.
78
What are Okazaki fragments, and how are they joined during DNA replication?
Okazaki fragments are short segments of DNA (100-1000 nucleotides long) synthesized on the lagging strand during DNA replication. They are joined together by DNA ligase, which catalyzes the formation of phosphodiester bonds between adjacent fragments after RNA primers are removed.
79
Describe the proposed mechanism for maintaining the rate and direction of DNA synthesis on both strands during replication
To maintain the rate and direction of DNA synthesis on both strands, it is postulated that the lagging strand loops back, allowing the lagging-strand polymerase to recycle from the end of one Okazaki fragment to the next RNA primer, forming a priming loop.
80
Explain the role of telomerase in solving the end-replication problem
The end-replication problem arises because the lagging strand cannot be fully replicated due to the absence of a template ahead of the replicating region for primase. Telomerase, a specialized reverse transcriptase, extends the telomere of the parental lagging strand by adding repeats using its RNA component (TERC), thereby providing room for primase to synthesize RNA primers.
81
What happens to the extreme ends of telomeric DNA in most adult somatic cells, and what are the consequences?
In most adult somatic cells, the extreme ends of telomeric DNA do not get replicated, leading to progressive telomere shortening. This phenomenon has been associated with aging.
82
How does DNA replication termination occur, and what enzymes are involved?
DNA replication termination occurs when the synthesized DNA strand meets the 5' end of another segment. RNA primers are removed by exonuclease I and RNAase H, and the resulting gaps between Okazaki fragments are filled in by DNA Polymerase δ. DNA ligase then joins the nicks between the synthesized fragments, completing replication.
83
What genetic diseases are associated with errors in DNA replication?
- Origin of replication defect - Meier-Gorlin syndrome, rare - characterised by very small ears & ear canals, short stature
- Helicase defect - Bloom Syndrome characterised by proportionate pre- and postnatal growth deficiency, sun-sensitive skin, predisposition to malignancy & chromosomal instability
- Polymerase defect: Hutchinson-Gilford progeria syndrome (HGPS) premature aging disorder
- POLE in somatic cancer: some colorectal and endometrial cancers
84
Define the stages of the cell cycle
The cell cycle consists of several stages:
Quiescent Phase (G0): A resting phase where the cell has left the cycle and stopped dividing.
Interphase: Divided into three sub-phases:
- G1 Phase: Growth phase where proteins and RNA are synthesized.
- S Phase: DNA synthesis occurs, replicating the genetic material.
- G2 Phase: Cell continues to grow, ensuring enough cytoplasmic materials for mitosis and cytokinesis.
Cell Division (M Phase): Involves nuclear division (mitosis) followed by cell division (cytokinesis).
85
Describe the regulatory mechanisms involved in the cell cycle
The cell cycle is regulated by checkpoints, which are regulatory pathways controlling the order and timing of cell cycle transitions. Cyclin-dependent kinases (CDKs) and cyclins form heterodimeric protein kinases, with CDKs acting as the catalytic subunit. Checkpoints ensure critical events are completed with high fidelity, halting the cell cycle if there is DNA damage or if processes such as DNA replication are incomplete.
86
Explain the significance of cell cycle checkpoints
Cell cycle checkpoints control the order and timing of cell cycle transitions, ensuring that critical events are completed accurately. They halt the cell cycle if DNA damage or incomplete processes such as DNA replication are detected, preventing the cell from progressing to the next stage until the issues are resolved
87
What are the three main cell cycle checkpoints?
G1/S (restriction) checkpoint
G2/M checkpoint
Metaphase / Spindle checkpoint
88
What is the G1/s checkpoint?
- 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
89
What is the G2/M checkpoint?
- 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)
- Allows G2-M phase transition
90
What is the metaphase/spindle checkpoint?
- Chromosomes assemble on metaphase plate
- Anaphase-promoting complex (APC) activated
- Degrades cyclin B = MPF disassembly
- Relieves inhibition of ‘separase’ which cuts cohesin
- Sister chromatid separation = anaphase entry
91
What is the role of p53?
p53 ("the guardian of the genome") plays an important role in controlling progression
through G1/S and G2/M checkpoints. p53 is a critical component of DNA damage checkpoints
92
How is the cell cucle used to produce high quality metaphase preps?
* 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.
93
Outline the process of meiosis and its stages
Meiosis is a specialized type of cell division that reduces the chromosome number by half. It involves two rounds of division (meiosis I and meiosis II). The stages of meiosis include:
Prophase I: Includes stages such as leptotene, zygotene, pachytene, diplotene, and diakinesis, involving chromosome pairing, crossing over, and recombination.
Metaphase I: Homologous chromosomes line up at the metaphase plate.
Anaphase I: Homologous chromosomes separate and move to opposite poles.
Telophase I and Cytokinesis: Nuclear membranes reform, and two haploid cells are formed.
Meiosis II: Similar to mitosis but with haploid cells as starting material, resulting in the formation of four daughter cells.
94
Describe the process of genetic recombination during meiosis
Genetic recombination occurs during prophase I of meiosis. Homologous chromosomes align and exchange segments of DNA through crossing over. This process involves the precise breakage and exchange of DNA segments between homologous chromosomes, leading to genetic diversity among the resulting daughter cells.
95
Where does transcription occur?
Transcription occurs in the nucleus, although mitochondrial DNA (mtDNA) genes are transcribed in the mitochondria.
96
How many classes of RNA polymerases are there, and which one is predominantly used for protein-coding genes?
There are three classes of RNA polymerases. The majority of protein-coding genes use RNA polymerase II.
97
Describe the stages of transcription
Transcription occurs in three stages:
Initiation
- Transcription initiation beings at the promoter region. Transcription factors bind to the promoter and position the RNA polymerase in order to initiate RNA synthesis- ‘basal transcription apparatus’
Elongation
- The template (antisense) DNA strand is a template for RNA polymerase
Termination
- Termination begins when a polyadenylation signal appears in the RNA transcript. The polyadenylation signal directs the cleavage of RNA polymerase.
98
What is the role of the TATA box in transcription?
The TATA box, typically located 25-35bp upstream of the transcriptional start site, defines the direction of transcription and indicates the DNA strand to be read.
99
What are enhancers and silencers in transcriptional regulation?
Enhancers are short DNA sequences that enhance gene expression, while silencers reduce transcriptional activity. They both bind to transcription factors and can be located at variable distances from the transcriptional start site.
100
What modifications occur during post-transcriptional processing of mRNA?
Post-transcriptional modifications include:
5’ capping
- Methylated nucleoside, 7-methylguanosine (m7G) is linked to the 5’ end of the RNA via a 5’-5’ phosphodiester bond
3’ polyadenylation
- AAUAAA is a polyadenylation sequence that signals 3’ cleavage forRNA Pol II transcripts (see above – ‘termination’).
- Cleavage of pre-mRNA occurs15-30 nucleotides downstream of AAUAAA signal.
- ~200 adenylate (AMP) residues are sequentially added by the enzyme poly(A)polymerase to form a poly(A) tail.
Splicing
- Splicing removes introns by endonucleolytic cleavage and ligation of exons
101
How does splicing occur in eukaryotes?
Splicing involves the removal of introns and ligation of exons, typically following 5’ capping and 3’ polyadenylation. It is sequence-dependent and mediated by a complex called the spliceosome
102
What is alternative splicing, and why is it significant?
Alternative splicing allows for the generation of multiple protein isoforms from a single gene. It contributes to proteomic diversity and regulates gene expression.
103
Outline the splicing mechanism
- It involves five snRNAs and their associated proteins. These ribonucleoproteins form a large (60S) complex, called spliceosome.
- After a two-step enzymatic reaction, the intron is removed and two neighbouring exons are joined together. The branch point A residue plays a critical role in the enzymatic reaction.
- Processivity of the spliceosome is largely progressive in a 5’ to 3’ direction in one intron (i.e. a 5’ splice site is recognised and then it scans until it meets a 3’ site)
- introns are not necessarily spliced out in order (e.g. intron 1, then intron 2); this is determined by RNA 20 structure and how accessible they are to the splicing machinery.
104
How is splicing regulating?
- Splicing is regulated by trans-acting elements and corresponding cis-acting elements (regulatory sites within the RNA)
- Tissue specific- cell cycle and other genes
105
How do mutations in splicing elements contribute to disease?
Mutations in splicing elements can lead to splicing abnormalities, which are implicated in various diseases, including cancer. Approximately 15–50% of human disease mutations affect splicing element
106
What are the stages of translation?
It occurs in three stages: initiation
- 5’ cap of mRNA recognised by the small ribosomal subunit – which then binds to mRNA and scans along the 5’UTR until start codon identified.
- Initiator tRNAMet (UAC) pairs with the complementary AUG start codon of the mRNA. Once this has successfully paired, the large unit of the ribosome attaches to the mRNA.
- Correct codon–anticodon pairing activates the GTPase centre of the ribosome
Elongation
- The ribosome must then shift in the 3' mRNA direction
Termination
- Elongation ends when a stop codon is reached (UAA, UAG, UGA). The tRNA complementary to the stop codon does not have an associated amino acid connected to it. This tRNA is known as the ‘release factor’
107
What role do ribosomes play in translation?
Ribosomes provide the structure for translation and catalyze the formation of peptide bonds between amino acids.
108
What is the role of tRNA in translation?
tRNA mediates the decoding of mRNA sequences to facilitate the incorporation of amino acids into polypeptide chains during translation.
109
What are regulatory elements in translation, and how do they function?
Regulatory elements, including cis-regulatory sequences, modulate translation efficiency by influencing mRNA stability and ribosome binding.
110
How do mutations in regulatory elements contribute to cancer?
Mutations in regulatory elements can dysregulate gene expression, leading to oncogenesis. Examples include mutations in promoter regions or alterations affecting mRNA processing in cancer-related genes.
e.g. Mutations in the promoter region of the TERT gene can lead to upregulation of telomerase expression in human cancer. Pathogenic hotspots are c.-146C>T (C250T) and c.-124C>T (C228T).
111
What is Nonstop Mediated decay (NSD), and what issue does it address in cells?
NSD is the detection and decay mechanism for mRNA transcripts lacking a stop codon. It addresses the issue of ribosomes stalling at the 3’poly-A tail region of transcripts without a stop codon, which prevents them from translating other mRNA molecules.
112
Describe the process of No-Go decay (NGD) and its targets.
NGD is a surveillance mechanism targeting mRNA transcripts where ribosomes have stalled during translation. It targets transcripts stalled by factors like strong secondary structures, hindering translational machinery movement.
113
What is Nonsense Mediated Decay (NMD), and what are its primary targets?
NMD targets transcripts containing premature termination codons (PTCs), protecting cells from deleterious truncated proteins. These PTCs can arise from various genetic and transcriptional errors.
114
How does NMD contribute to the quality control of the cell's transcriptome?
NMD targets non-functional pseudogenes, mRNAs with reading frames in the 5’UTR, and those escaping nuclear retention. It regulates physiological mRNAs involved in various cellular processes like stress responses, neuronal development, and DNA repair.
115
Explain the molecular mechanism of NMD involving major components like UPF1, UPF2, UPF3, and SMG proteins.
UPF1 interacts with UPF2 and UPF3 upon recognizing a stop codon, triggering phosphorylation and recruitment of SMG proteins. This forms a complex that leads to mRNA degradation
116
What are some exceptions to the NMD target rule regarding the location of premature termination codons (PTCs)?
Exceptions include transcripts with PTCs far upstream of a poly(A) tail, long 3’ UTRs, and certain gene rearrangements, which may still be targeted by NMD despite PTC location.
117
How does NMD contribute to disease, and provide examples of diseases influenced by NMD?
NMD can aggravate or mitigate disease severity depending on factors like mRNA stability. Diseases like β-thalassemia, Duchenne muscular dystrophy (DMD), and cancer are influenced by NMD.
118
Name some functional non-coding RNAs
Ribosomal RNA (rRNA)
Small nuclear RNA (snRNA)
Small Nucleolar RNA (snoRNA)
Transfer RNA (tRNA)
119
What are the components of cytoplasmic ribosomes?
Cytoplasmic ribosomes consist of two subunits: the larger 60S subunit and the smaller 40S subunit. The 60S subunit contains 28S, 5.8S, and 5S rRNA molecules, while the 40S subunit contains 18S rRNA.
120
What is the pathogenic mechanism associated with ribosomopathies?
Pathogenicity in ribosomopathies is thought to result from an accumulation of free ribosomal proteins that bind the p53 suppressor MDM2, leading to activation of p53 and increased apoptosis and cell death.
121
Name two ribosomopathies caused by defects in ribosomal proteins and their associated genes
Examples include 5q- syndrome, associated with haploinsufficiency of RP514, and Diamond Blackfan anemia (DBA), mainly caused by haploinsufficiency of the gene encoding RPS19
122
What is the primary role of small nuclear RNAs (snRNAs)?
snRNAs primarily participate in the formation of the spliceosome, which is involved in pre-mRNA splicing. There are nine human spliceosomal snRNAs.
- Sm-class RNAs
- Lsm-class RNAs
123
What is the primary function of snoRNAs?
snoRNAs mainly function in RNA modification and processing.
124
How are snoRNAs involved in cancer, according to recent studies?
Certain snoRNAs have been implicated in cancer, such as U50 in non-small cell lung cancer, suggesting a potential role in tumorigenesis.
125
What is the primary function of transfer RNAs (tRNAs)?
tRNAs transfer the correct amino acid to the ribosome during protein synthesis.
126
What is wobble base pairing, and how does it contribute to tRNA function?
Wobble base pairing allows the third base of a tRNA anticodon to form hydrogen bonds with multiple bases at the 3’ position of a codon, increasing the versatility of tRNA recognition.
127
Give examples of normal variation in the human genome
Heteromorphs
Copy number polymorphism
Transposable elements
Fragile sites
SNPs
128
What are heteromorphs in the context of genomic variation?
Heteromorphs are microscopically visible variations observed in the human genome, often involving heterochromatin or euchromatic variants, differing in size, morphology, or staining properties.
129
Define copy number polymorphism.
Copy number polymorphism refers to segments of DNA present at a variable copy number compared to a reference genome, regardless of their pathogenicity. They include aneuploidies, large deletions, duplications, and insertions, which can be visible at a microscopic level or identified through molecular screening methods.
130
What are transposable elements, and how do they contribute to genomic variation?
Transposable elements (TEs) are units of DNA that can move within the genome, either by replicating themselves (retrotransposons) or by transposing directly without replication (DNA transposons). As they accumulate over evolutionary time, they create repetitive sequences of DNA interspersed throughout the genome, potentially affecting gene expression and contributing to genetic diversity.
131
Explain the significance of fragile sites in the genome
Fragile sites are segments of uncoiled chromatin that are prone to gaps and breaks in chromosomes. They are classified as rare, intermediate, or common, and while most are normal variants, some coincide with cancer breakpoints or are associated with pathological changes, such as mental retardation.
132
What is the main characteristic of single nucleotide polymorphisms (SNPs)?
SNPs involve variants in a single nucleotide base, occurring with a frequency of 1 in every 100-300 nucleotides. They are the most abundant type of genetic variation, often found in non-coding regions, and can mark alternative ancestral chromosome segments.
133
What are the sources of DNA damage?
DNA damage can arise from either endogenous sources, such as internal chemical events like depurination and oxidative damage, or exogenous factors like mutagenic chemicals (e.g., tobacco smoke) and certain types of radiation (UV and ionizing).
134
What are the potential effects of point mutations on protein coding regions?
Point mutations can lead to alterations in protein coding regions, potentially impacting protein structure or function.
135
Describe recombination and its potential outcomes
Recombination involves the restructuring of part of a genome, which may result in chromosome abnormalities such as deletions, duplications, translocations, and inversions.
136
Name the three mechanisms of mutations in DNA.
The three mechanisms of mutations in DNA are DNA damage, errors in DNA replication or recombination, and failure to repair DNA damage.
137
What are the two categories of DNA repair mechanisms?
DNA repair mechanisms include direct reversal of the chemical process generating the damage and the replacement of damaged nucleotide bases.
138
Describe the process of base excision repair (BER).
Base excision repair corrects DNA damage from oxidation, deamination, and alkylation by excising the damaged base and filling in the resulting gap with DNA polymerase.
139
What is nucleotide excision repair (NER) responsible for addressing?
This repair pathway consists of 4 steps; (1) Detection of damage > (2) Nuclease excision of the section of DNA that includes and surrounds the error > (3) Filling in of the resulting gap by DNA polymerase > (4) Sealing the nick between newly synthesised and older DNA
140
What is the primary purpose of mismatch repair (MMR)?
Mismatch repair (MMR) counteracts replication errors by resolving single nucleotide mismatches and small insertion loops generated during DNA replication.
Upon recognition of a mismatch, MutSα or MutSβ recruits the MutL (heterodimer MLH1-PMS2] complex. The mismatched base(s) are excised. DNA polymerase fills in the gap, following which the strands are sealed by the action of a DNA ligase.
When MMR is lost or defective there is a decrease in apoptosis, an increase in cell survival, and a potential increase in damage-induced mutagenesis.
141
What are the main mechanisms for repairing double-strand breaks (DSBs)?
The main mechanisms for repairing DSBs are homologous recombination (HR) and non-homologous end joining (NHEJ).
142
Explain the difference between HR and NHEJ
HR utilizes homologous DNA sequences for precise repair and is limited to the S/G2 phases of the cell cycle, while NHEJ directly joins DNA ends and operates throughout the cell cycle.
143
What is replication slippage, and what types of mutations can it lead to?
Replication slippage is a mutation that results in trinucleotide or dinucleotide expansions or contractions during DNA replication, often associated with tandem repeats.
144
What is homologous recombination repair deficiency (HRD), and what are its implications in cancer?
HRD is a common feature of high-grade serous ovarian cancers, often caused by mutations in DNA repair genes such as BRCA1 and BRCA2, and it can affect the response to PARP inhibitor therapy.
145
What disease is associated with MMR deficiency?
Lynch syndrome
- All colorectal cancers get testing
- Using immunohistochemistry for mismatch repair proteins or microsatellite instability testing to identify tumours with deficient DNA mismatch repair, and to guide further sequential testing for Lynch syndrome
146
What are pseudogenes, and what is their typical characteristic?
Pseudogenes are redundant copies of protein-coding genes with high sequence homology to parent genes. They are often considered dysfunctional due to the accumulation of mutations, including premature stop codons, frameshifts, or indels.
147
How are pseudogenes categorized?
Pseudogenes are categorized into three primary classes: processed, unprocessed (or duplicated), and unitary pseudogenes.
148
What are some mechanisms by which pseudogene transcripts can control the expression of their parent genes?
Pseudogene transcripts can act as competitive endogenous RNAs (ceRNAs), antisense transcripts, precursors for small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs).
149
Provide an example of a pseudogene affecting the expression of its parent gene and its impact on cancer
The pseudogene FLT1P1 produces sense and antisense transcripts that reduce the levels of the parent gene VEGFR1, thereby decreasing human colorectal tumor cell proliferation and xenograft tumor growth
150
How can pseudogenes regulate their parent genes through competitive endogenous RNA (ceRNA) mechanisms?
Pseudogenes can regulate their parent genes by acting as decoys to absorb microRNAs targeting the parent gene for degradation, thus upregulating the expression of the parent gene.
151
What is the role of PTENP1 in cancer, and how does it regulate its parent gene PTEN?
PTENP1 regulates its parent gene PTEN through a ceRNA mechanism, functioning as a decoy to absorb microRNAs targeting PTEN for degradation. This upregulation of PTEN suppresses tumor growth.
152
How can processed pseudogenes impact the genome through coding-dependent mechanisms?
Although processed pseudogenes are not compatible with protein-coding capacity by definition, some have undisrupted open reading frames (ORFs) that are translated.
153
What is the clinical significance of pseudogenes in genetic testing?
Pseudogenes can complicate mutation screening by causing false results, particularly in Sanger sequencing methods. Amplification strategies specific to the parental gene are necessary to avoid this issue.
154
How can the presence of pseudogenes affect next-generation sequencing pipelines?
Pseudogenes need to be considered in next-generation sequencing pipelines to accurately differentiate pseudogenes from their parent mRNAs and avoid misinterpretation of results.
155
What is the potential application of pseudogenes in cancer research and clinical practice?
Pseudogenes have potential applications as prognostic and diagnostic markers, as well as in stratifying patient subtypes in multiple cancers.
156
Provide an example of a pseudogene affecting cancer progression and its associated parent gene.
PTENP1 activity has been observed in various cancer types, including Oesophageal Squamous Cell Carcinoma (SCC), oral SCC, head and neck SCC, and melanoma, where it sensitizes cells to specific treatments and impacts tumor growth.
157
How do pseudogenes like BRAFP1 contribute to oncogenesis?
BRAFP1, a pseudogene of BRAF, plays an oncogenic role by activating the MAP kinase pathway, leading to the formation of tumors, and is frequently found to be aberrantly expressed in cancers such as B cell lymphomas.
158
What challenges do pseudogenes pose in mutation screening, and how can these challenges be addressed?
Pseudogenes pose challenges in mutation screening due to their high similarity to parent genes, leading to potential false results. Strategies such as long-range PCR and specific primer design are employed to ensure amplification of the parental gene only.
159
What role do pseudogenes play in the evolution of the genome, particularly in terms of enhancer-like elements?
Pseudogenes can evolve proto-promoter activity and influence proximal genes by containing transcription factor-binding motifs (TFBMs), potentially evolving into enhancer-like elements and impacting neighboring genes.
160
What are clastogens, and what types of agents can induce double-strand breaks (DSBs) in DNA?
Clastogens are endogenous chromosome breaking agents. Exposure to exogenous agents such as ionizing radiation can also produce DSBs in DNA.
161
What are the potential outcomes when DSBs interact with other DSBs?
Interaction between DSBs can result in structural abnormalities such as rearrangements in the genome.
162
Describe the role of non-allelic homologous recombination (NAHR) in recurrent rearrangements.
NAHR occurs between low copy repeats (LCRs) or other repetitive elements, serving as substrates and essential requirements for recurrent rearrangements.
163
How does the breakage-fusion-bridge cycle contribute to chromosome instability in cancer?
This cycle involves chromosome or sister-chromatid fusion due to telomere erosion, leading to dicentric chromosomes, breakage, and further rounds of fusion and breakage. It contributes to chromosome instability in cancer.
164
What are some factors influencing partner choice in reciprocal translocations?
Factors include spatial proximity within the nucleus, the frequency and likelihood of a locus harboring a double-strand break, and the activity of enzymes like activation-induced cytidine deaminase (AID).
165
Describe the formation of ring chromosomes and the proposed mechanisms of repair.
Ring chromosomes result from terminal breaks in both chromosome arms, followed by fusion of the broken ends. Repair mechanisms may include NHEJ or MMBIR.
166
What is chromothripsis, and what are its potential underlying mechanisms?
Chromothripsis involves massive genomic rearrangements that occur as a one-off event. Potential mechanisms include double-strand breaks in micronuclei and DNA replication stress.
167
Explain the formation of isodicentric chromosomes and the mechanisms involved
Isodicentric chromosomes can result from a U-type exchange between low copy repeats (LCRs) on homologs. Mechanisms may also involve NAHR or replication-based mechanisms.
168
Describe the formation of Robertsonian translocations and the mechanisms involved.
Robertsonian translocations can result from centric fusion, breakage in one short and one long arm, or misdivision of the centromere. NAHR between olfactory receptor genes on different chromosomes can also contribute.
169
What are some mechanisms of terminal deletions, and how are they stabilized?
Terminal deletions can be stabilized by telomere healing, telomere capture, or chromosomal circularization. Stabilization may involve the presence of repetitive elements.
170
Explain the formation of reciprocal translocations and the role of double-strand breaks.
Reciprocal translocations can be formed via mechanisms such as NHEJ, MMEJ, FoSTeS, or MMBIR. Double-strand breaks play a crucial role in initiating these rearrangements.
171
What is the concept of genome chaos, and how does it relate to cancer evolution?
Genome chaos involves massive genomic rearrangements and contributes to cancer evolution, characterized by punctuated macroevolution at the genome level and stepwise microevolution at the gene level.
172
What are epigenetic changes, and how do they differ from changes in the primary DNA sequence?
Epigenetic changes are heritable and transient alterations in gene expression that do not alter the primary DNA sequence. They control gene expression by switching genes on/off or modulating their expression levels, contributing to variable expression in different cell types.
173
Name three mechanisms of epigenetic gene regulation
Three mechanisms of epigenetic gene regulation are histone modification, non-coding RNA regulation, and DNA methylation
174
How is DNA methylated, and what is its significance in gene regulation?
DNA methylation involves the addition of a methyl group (CH3) to cytosine, usually in CpG dinucleotides, by DNA methyltransferase enzymes. Methylation is associated with gene silencing and is essential for imprinting, X-chromosome inactivation, and genome stability
175
Describe the role of DNA methyltransferases (DNMTs) in DNA methylation
DNMT enzymes include DNMT1, which maintains methylation patterns during DNA replication, and DNMT3A and DNMT3B, which establish de novo methylation patterns. TET enzymes are involved in active demethylation by converting 5-methylcytosine (5MeC) to 5-hydroxymethylcytosine (5hmC)
176
What are MeCpG-binding proteins, and what is their role in epigenetic regulation?
MeCpG-binding proteins recognize methylated DNA and recruit other proteins associated with repressive structures such as histone deacetylases (HDACs). They regulate chromatin structure and gene expression. Examples include MBD1-4 and MECP2, mutations in which can cause Rett syndrome.
177
How do epigenetic changes contribute to cancer development?
Epigenetic changes in cancer cells include global loss of DNA methylation, leading to abnormal gene activation, and hypermethylation of CpG islands, silencing tumor suppressor genes. These changes contribute to tumorigenesis by promoting genomic instability, reactivating transposable elements, and disrupting imprinting.
178
What is microsatellite instability, and how is it related to epigenetic changes in cancer?
Microsatellite instability is indicative of a defect in DNA repair genes, particularly the mismatch repair genes, and can be caused by hypermethylation of the promoter of the DNA repair gene MLH1. However, MLH1 promoter methylation is a sporadic event and not consistent with a diagnosis of Lynch syndrome.
179
How does epigenetics relate to complex diseases other than cancer?
Epigenetic changes are implicated in the expression of common complex diseases such as asthma, schizophrenia, and inflammatory bowel disease. They contribute to differential gene expression and disease susceptibility.
180
Describe epigenetic therapy and its potential applications.
Epigenetic therapy aims to alter DNA methylation or histone acetylation patterns to treat diseases. Drugs such as DNA methyltransferase inhibitors and HDAC inhibitors have shown promise in reactivating silenced genes in cancer and other disorders. However, caution is needed to avoid off-target effects on normal cells.
