euk-prok + cancer

Cell size (euk vs prok)

Eukaryotic cells are larger, 10-100µm in diameter, while prokaryotic cells are smaller, 0.5-5µm in diameter

Nucleus (euk vs prok)

Eukaryotic cells have a nucleus with nuclear envelope present while prokaryotic cells have no true nucleus / no nuclear envelope

Genetic material (euk vs prok)

Eukaryotic cells have linear DNA associated with histone proteins, found in a membrane bound nucleus, with no plasmids, while prokaryotic cells have circular DNA associated with few histone-like proteins, found in a region of the cytoplasm known as the nucleoid region, with plasmids present

Ribosome for protein synthesis (euk vs prok)

Eukaryotic cells have 80S ribosomes which may be attached to ER or float freely in cytoplasm, while prokaryotic cells have 70S ribosomes, have no ER present, and ribosomes float freely in cytoplasm

Organelles (euk vs prok)

Eukaryotic cells have many membrane bound organelles present while prokaryotic cells have no membrane bound organelles

Cell walls (euk vs prok)

Eukaryotic cell walls are composed of cellulose in plants & chitin in fungi while prokaryotic cell walls are composed of peptidoglycan

Size of genome (euk vs prok)

The eukaryotic genome is 10^7-10^11 base pairs (larger) while the prokaryotic genome is 10^4-10^7 base pairs (smaller)

Appearance of genome (euk vs prok)

The eukaryotic genome consists of multiple, linear molecules while the prokaryotic genome is generally a single, circular molecule

Molecule (euk vs prok)

Both eukaryotic and prokaryotic genomes are double helix DNA

Association with proteins (euk vs prok)

The eukaryotic genome has large amounts of association with proteins e.g. histones, scaffold proteins, while the prokaryotic genome has relatively less histone-like proteins

Level of DNA packing/coiling & pathway (euk vs prok)

Eukaryotic DNA packing is high

Location (euk vs prok)

The eukaryotic genome is located in the nucleus while the prokaryotic genome is located in the nucleoid region – not membrane-bound

Extrachromosomal DNA (euk vs prok)

Eukaryotic cells have extrachromosomal DNA if you consider mitochondrial and chloroplast circular DNA, while prokaryotic cells have plasmids (much smaller rings of DNA) as extrachromosomal DNA

Number of genes (euk vs prok)

Eukaryotic cells have 25,000 genes while prokaryotic cells have 4,500 genes

Non-coding regions (euk vs prok)

Non-coding regions are common in the eukaryotic genome – about 98%, while they are not common in the prokaryotic genome – typically less than 15%

Presence of introns (euk vs prok)

Introns are many in the eukaryotic genome while they are rare in the prokaryotic genome

Presence of promoters (euk vs prok)

Promoters are present in both eukaryotic and prokaryotic genomes

Presence of repeated sequences (euk vs prok)

Repeated sequences are many in the eukaryotic genome (e.g. telomeres & centromeres) while they are few in the prokaryotic genome

Presence of enhancers/silencers (euk vs prok)

Enhancers/silencers are common in the eukaryotic genome while they are rare in the prokaryotic genome

Presence of operons (euk vs prok)

Operons are very few known in the eukaryotic genome while they are many in the prokaryotic genome

Note on packing of eukaryotic DNA

The packing of the very long eukaryotic DNA ensures that the DNA is compact & fits into the nucleus and that the DNA does not get entangled and break

Telomerase mechanism

A short 3-nucleotide segment of RNA within telomerase binds to part of a DNA repeat in the 3'overhang by complementary base pairing → the adjacent part of the RNA within telomerase is used as a template to synthesise a short complementary 6-nucleotide DNA repeat → telomerase catalyses the formation of the phosphodiester bonds between the existing 3'OH group of existing DNA overhang and 5' phosphate group of incoming deoxyribonucleotide → telomerase translocates 6 nucleotides to the right in the 5' to 3' direction of the DNA overhang and begins to make another repeat, this process being repeated to make a series of tandem repeats, elongating the telomere → then primase makes an RNA primer near the end of the telomere, DNA polymerase adds nucleotides to the 3'OH end of the primer and synthesizes a complementary strand, the nick is sealed by ligase, and the RNA primer is eventually removed

Introns

non coding DNA sequences found within a gene, specifically between exons in a specific segment of DNA (also present in pre-mRNA), only in eukaryotes. They enable alternative RNA splicing to occur where a single pre-mRNA can have all its introns and different combinations of its exons excised and the remaining exons joined such that different mature mRNAs are produced, so one gene can code for more than one polypeptide

Promoter

located just upstream of the transcription start site of a gene → hence called a proximal control element, has critical elements e.g.1. TATA box (located upstream of transcription start site) e.g. 2. CAAT and GC boxes (located upstream of TATA box but not always present). It is the recognition & binding site for general transcription factors which then recruit RNA polymerase to form transcription initiation complex which initiates transcription. TATA box in promoter determines precise location of transcription start site. CAAT and GC box improve efficiency of promoter

Enhancer

usually located far away from the promoter (much further upstream or downstream) → hence called a distal control element. It is the recognition & binding site for activators (specific transcription factors) and increases the frequency of transcription by promoting the assembly of the transcription initiation complex

Silencer

usually located far away from the promoter (much further upstream or downstream) → hence called a distal control element. It is the recognition & binding site for repressors (specific transcription factors) and decreases the frequency of transcription by preventing the assembly of the transcription initiation complex

Note on activators and repressors

Activators and repressors are not enzymes & do not have an active site. Instead, each has a DNA binding site that is complementary in shape & charge to a specific regulatory sequence of DNA which it binds to (e.g. enhancer/silencer)

Telomere

found at both ends/terminals of linear, eukaryotic chromosomes, non-coding DNA made up of a series of tandem repeat sequences (a specific sequence of nucleotides occurring many times in a row), in humans each repeat has the sequence 5' TTAGGG 3', and has a single stranded region at their 3' ends known as the 3' overhang (due to a limitation of DNA polymerase, this region of DNA does not have a complementary strand). Telomeres ensure that genes are not eroded and vital genetic information is not lost with each round of DNA replication due to the end replication problem, since telomeres are non-coding sequences that will be lost before any vital genetic information is, and can be lost without any deleterious effect. Telomeres protect and stabilize the terminal ends of chromosomes by forming a loop using the 3'overhang, preventing single-stranded terminal ends of chromosomes from annealing to each other (preventing fusing of chromosomes) and preventing the cell's DNA repair machinery from detecting the chromosome as damaged DNA and triggering apoptosis. Telomeres also allow their own extension, as the 3' overhang provides an attachment point for the enzyme telomerase; although telomeres shorten with every round of DNA replication, telomerase activity in germ cells, embryonic stem cells and cancer cells can maintain telomere length

Centromere

constricted region on chromosome where kinetochore microtubules attach during nuclear division, non-coding DNA made up of a series of tandem repeat sequences. It allows sister chromatids to adhere to each other, allows kinetochore proteins to attach and in turn allow spindle fibres to attach so that sister chromatids/homologous chromosomes can align along the metaphase plate and be separated to opposite poles, ultimately allowing proper alignment and segregation of chromosomes

Cellular differentiation

in a multicellular organism, all somatic cells carry identical genes but cells show a wide variation in structure and function due to the different proteins that they express

Adapt to changes

the kinds of proteins produced by an organism varies according to circumstances and demand

Conserve resources

transcriptional level of control predominates as it is the most efficient mechanism with minimal wastage, especially in prokaryotes

More varied proteome despite limited genome size

by alternative splicing

Histone acetylation vs deacetylation

Histone acetylation is the addition of acetyl group to lysine residues on the histone by histone acetyltransferase/histone acetylase → removes +ve charge on histones → decreases electrostatic attraction between –vely charged DNA and histones → promoter region is more accessible to RNA polymerase and general transcription factors → promotes transcription as it promotes assembly of transcription initiation complex. Histone deacetylation is the removal of acetyl groups from histones by histone deacetylase → restores +ve charge on histones → restores tighter interaction between DNA & histones → promoter region is less accessible → inhibits transcription as it prevents assembly of transcription initiation complex

Chromatin remodeling complex

protein complexes which alter structure of nucleosomes temporarily. They can cause DNA to be more tightly coiled around histones and prevent access of RNA polymerase and general transcription factors to promoter → inhibits transcription, or can cause DNA to be less tightly coiled around histones and give access to RNA polymerase and general transcription factors to promoter → promotes transcription

DNA methylation

addition of a methyl group by DNA methylases to selected cytosine residues in CG sequences, prevents transcription/causes gene silencing by (1) blocking the binding of transcription factors at the promoter and hence preventing the formation of the transcription initiation complex, and (2) recruiting DNA-binding proteins (e.g. transcriptional repressors, histone deacetylases and repressive chromatin remodeling complexes)

Activators vs repressors (transcription level)

Activators bind to enhancers → promote assembly of transcription initiation complex as bending of spacer DNA allows interaction of activators with RNA polymerase and/or GTFs at the promoter → transcription frequency increases (may recruit histone acetyltransferase and chromatin remodeling complex to decondense chromatin). Repressors bind to silencers → prevent assembly of transcription initiation complex as bending of spacer DNA allows repressor to bind to general transcription factors, preventing activators from binding → transcription frequency decreases (may recruit histone deacetylase and repressive chromatin remodeling complex)

Addition of 5' cap

Addition of a 7-methylguanosine nucleotide to the 5' end of the pre-mRNA, occurring shortly after transcription begins (co-transcriptionally). It helps the cell recognize mRNA so subsequent steps like splicing and polyadenylation can occur, acts as a signal to export mRNA out of nucleus, and stabilises and protects the growing pre-mRNA chain from degradation by ribonucleases

Intron splicing & alternative splicing

Introns (noncoding regions within a gene) are excised and exons (coding regions) are joined together by spliceosomes (a complex of snRNA and proteins

Addition of poly A tail (polyadenylation)

The 3'end of a pre-mRNA is cleaved by an endonuclease downstream of the polyadenylation signal (AAUAAA), then a poly-A polymerase adds a long sequence of adenine nucleotides to the 3' end, forming a poly(A) tail, occurring immediately after transcription. It acts as a signal to export mature mRNA out of nucleus through nuclear pores, stabilizes and protects the mature mRNA from degradation by ribonucleases, and interacts with eukaryotic initiation factors and the 5'cap for initiation of translation

mRNA half-life

determined by the length of its poly-A tail; the longer the poly-A tail, the longer the time mRNA can be used as a template to make proteins. The poly-A tail is removed by ribonucleases in the 3' to 5' direction until a critical length is reached which triggers removal of the 5'cap and degradation of the mRNA from the 5'end too

Anti-sense RNA

Anti-sense RNAs complementary to part of the mRNA to be degraded will bind to the mRNA; this double stranded RNA will block translation of the mRNA and be targeted for degradation by nucleases

Formation of translation initiation complex

During translation initiation, small ribosomal subunit, eukaryotic initiation factors and initiator tRNA form a complex which binds to the 5'cap and the 3'poly A tail causing the mRNA to circularise; this can be prevented by binding of translational repressor to the 5'cap, 5'UTR, or 3'UTR, which interferes with the interactions needed for translation initiation

Availability of activated eukaryotic translation initiation factors

eukaryotic initiation factors facilitate the binding of the small ribosomal subunit to the 5'cap; their availability is determined by whether or not they are phosphorylated (some activated by phosphorylation, others inactivated), and without activated initiation factors, translation cannot begin

Formation of functional proteins

Covalent modification/cleavage (e.g. attachment of prosthetic groups, glycosylation, disulfide bond formation) of polypeptides make them functional proteins

Regulation of protein activity

Phosphorylation/dephosphorylation of eukaryotic translation initiation factors can activate/deactivate the protein and hence up/down regulate its activity

Protein degradation

Protein degradation by proteasomes determines how long a protein remains in a cell to carry out its function; proteins targeted for degradation are tagged with ubiquitin (by ubiquitin ligase) so they can be recognised by proteasomes, where they are degraded into peptides while the ubiquitin is recycled

Note on regulation levels

1-3 is regulation at genomic level, 4-5 is regulation at transcriptional level, 6-7 is regulation at post-transcriptional level, 8-11 is regulation at translation level, and 12 is regulation at post-translational level

Environmental factors

exposure to carcinogens (e.g. tar in cigarette smoke, asbestos etc.) and ionizing radiation (e.g. uv radiation, X-rays) can cause mutations that can lead to cancer

Loss of immunity due to infection with certain viruses

HIV can weaken the immune system and reduces the body's ability to fight infections by other viruses (e.g. Kaposi's sarcoma-associated Herpes virus) that can cause cancer

Genetic predisposition

due to gene mutations (in the germline cells) which we inherit from our parents (e.g. BRCA1 gene, a tumour suppressor gene)

Age

chances of getting cancer increases with age due to accumulation of mutations in a cell over a lifetime

Proto-oncogenes

code for proteins (e.g. growth factors, activators, growth signal transduction factors) that stimulate normal cell division/proliferation; when mutated, they are known as oncogenes. Oncogenes can form by (a) increasing the amount of proto-oncogene protein product via (i) point mutation in regulatory elements creating a stronger promoter → increased transcription frequency and excess protein product, (ii) gene amplification, where the number of copies of a proto-oncogene is increased due to a DNA replication mistake, (iii) chromosomal translocation placing the proto-oncogene under control of an enhancer, or (iv) retroviral integration which can inactivate a silencer, insert a viral enhancer, or insert a viral homologue of the proto-oncogene — all leading to excessive protein product and uncontrolled cell division; or by (b) increasing the intrinsic activity of the protein product via a point mutation within the proto-oncogene, changing the amino acid sequence so the protein becomes hyperactive or more resistant to degradation, leading to uncontrolled cell division. Example

Tumour suppressor genes

code for protein products that inhibit cell division and prevent uncontrolled cell division by upregulating genes involved in cell cycle arrest, DNA repair and/or apoptosis; when mutated (in promoter or coding sequence), they are inactivated, and both copies of the allele need to be mutated for the effect to be observed. Example

Gain-in-function vs loss-of-function mutation

A gain-in-function mutation (e.g. proto-oncogene like ras mutated to form an oncogene) is dominant — mutation in just one allele can result in uncontrolled cell division due to increased synthesis/activity of a functional product not produced previously. A loss-of-function mutation (e.g. tumour suppressor genes like p53) is recessive — mutations in both copies of the allele are necessary for the phenotype to be observed, since the non-mutant copy still produces a functional gene product and masks the effect of the mutant copy

Why cancer is a multi-step process

The development of cancer requires the accumulation of mutations in genes controlling regulatory checkpoints of the cell cycle in a single cell, disrupting the normal cell cycle and causing excessive cell proliferation. A gain-in-function mutation in a proto-oncogene results in overexpression, producing excessive/hyperactive growth factors leading to excessive cell proliferation, while a loss-of-function mutation in both alleles of a tumour suppressor gene disrupts the cell's ability to inhibit the cell cycle, enable DNA repair, and promote apoptosis. Activation of genes coding for telomerase lengthens telomeres, allowing the cell to divide indefinitely and accumulate more mutations. Loss of contact inhibition enables cells to grow into a benign tumour; angiogenesis within the tumour transports oxygen and nutrients for growth; the presence of blood vessels can then result in a malignant tumour capable of metastasising via the bloodstream to form secondary tumours. As it takes years to accumulate these mutations, the chances of developing cancer increases with age

Checkpoints and dysregulation

Not all cells need to be replaced at the same rate or time; there are built-in controls in the cell cycle (checkpoints) that ensure normal cell division, and dysregulation of these checkpoints can result in uncontrolled cell division leading to tumour formation and eventually cancer

Nuclei (normal vs cancer)

Normal cells have normal nuclei with low nucleo-cytoplasmic ratio while cancerous cells have abnormal nuclei with high nucleo-cytoplasmic ratio

Genes (normal vs cancer)

Normal cells have proto-oncogenes and tumour suppressor genes not mutated while cancerous cells have oncogenes and mutated tumour suppressor genes present

Cells (normal vs cancer)

Normal cells have regular shape while cancerous cells vary in shape and size

Cell division (normal vs cancer)

Normal cells undergo controlled cell division & proliferation while cancerous cells undergo excessive uncontrolled cell division & proliferation

Apoptosis (normal vs cancer)

Normal cells undergo apoptosis, dividing for a certain number of times and then stop dividing, while cancerous cells do not undergo apoptosis and can divide indefinitely

Contact inhibition (normal vs cancer)

Normal cells show contact inhibition, not dividing further when in contact with other cells → forming a monolayer of cells, while cancerous cells do not show contact inhibition → forming multiple layers of cells

Differentiation (normal vs cancer)

Normal cells can differentiate to become specialized cells while cancerous cells do not differentiate

Cell adhesion/metastasis (normal vs cancer)

Normal cells show cell adhesion → formation of tissues and organs, while cancerous cells can detach from surrounding cells and metastasize

Angiogenesis (normal vs cancer)

Normal cells do not stimulate new blood vessels while cancerous cells stimulate growth of new blood vessels