Basic Molecular Biology Flashcards
What is the structure of DNA?
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).
What is the structure of RNA?
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
How does DNA form a double helix?
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
What are the different conformations of DNA?
B-DNA
A-DNA
Z-DNA
Cruciform and Hairpin Structures
H-DNA
G4-DNA
What is B-DNA?
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.
What confirmation does RNA have?
A-DNA: right-handed helix with 11 base pairs per turn. RNA adopts A form. Not found in vivo (DNA)
What are histones?
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
What is a nucleosome?
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
What are chromatin?
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.
What are euchromatin?
Euchromatin: relatively extended conformation “open” DNA under active transcription. Marked by weak binding of H1 histones and acetylation of the histones.
What are heterochromatin?
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
What is CTCF?
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.
What are Topologically associated domains (TADs) ?
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
What is histone modification?
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
What is histone acetylation?
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
What regulates histone acetylation?
Regulated by the opposing action of histone acetyl-transferases (HATs) and histone deacetylases (HDACs)
What is histone phosphorylation?
Occurs on Serines, Threonines and Tyrosines.
Addition of phosphate to amino acid adds significant negative charge to the histone, influencing chromatin structure
Phosphorylation of H3S10 during mitosis occurs genome-wide - associated with chromatin condensing
What regulates histone phosphorylation?
Controlled by kinases (adds phosphates) and phosphatases (removes phosphates).
What is histone methylation?
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)
What regulates histone methylation?
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.
What other histone modifications are there?
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
What are chromatin remodellers?
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.
What are the 5 families of chromatin remodellers?
SWI/SNF
ISWI
NuRD/ Mi-2 CHD
INO80
SWR1
What is the role of DNA methylation in cancer development by influencing gene expression?
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
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.
What are the four types of chromosomes?
Metacentric
Submetacentric
Acrocentric
Telocentric (only centromere and long arms- not in humans)
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.
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.
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.
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.
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.
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.
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.
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-
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.
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.
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.
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.
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.
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.
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
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.
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).
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.
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.
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
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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)
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.
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.
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).
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
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.
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.
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
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
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.
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.
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.
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
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
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.
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.
