Cells to Organisms

Block 1- Introduction to Prokaryotes and Eukaryotes

What are the 3 main domains of life

Eukarya, Archea, and Bacteria

What are the differences between prokaryotes and eukaryotes

Eukaryotes

• Larger

• Compartmentalisation

• Nucleus

Prokaryotes

• Smaller

• No compartmentalisation

• No nucleus

What are the 3 main forms of prokaryote

• Coccus

• Rod/Bacillus

• Sprillum/Spirochete

Describe prokaryote cell structure

What is the difference between gram positive and gram negative

Named after the ability to take up a gram stain

Gram positive- turns purple

Gram negative- turns pink

How does the structure of the outer membrane differ in gram negative and gram positive bacteria.

The gram stain wont stick to the gram -ve due to the outer membrane. Peptidoglycan is made out of alternating sugars and amino acids.

What are some functions of the prokaryotic membrane

• Permeability barrier

• Protein anchor

• Energy conservation

• Pumps control permeability

• Membrane uses proton motive force for ATP production

How is genetic material organised in prokaryotes

Prokaryote chromasomes are circular, supercoiled and there is only one copy. Plasmids carry accessory genetic material used in horizontal gene transfer

Describe prokaryotic ribosomes

• 70s size (smaller than eukaryotes)

• Due to lack of compartmentalisation, transcription and translation are coupled

• mRNA : RNAP : Ribosome complex - multiple ribosomes get loaded onto a single mRNA (polysome)

What is the glycocalyx / EPS

• A sticky/slimy coat made up of polysaccharides, proteins and glycoproteins / glycolipids

• EPS stands for extracellular polymeric substance

• Subdivided into capsules and slime layers

What are the differences between capsules and slime layers

Capsules - layer organized into a tight matrix with excludes small particles

Slime layers- Easily deformed and loosly attached

What is the role of capsules/slime layers

• Adherence of bacteria to surfaces

• Formation of biofilms

• Capsule acts as a virulence factor

• Helps bacteria avoid destruction by the hosts immune system

What are fimbrae

Plays a role in adherence of bacteria to host cells in symbiotic or pathogenic situations

What are Pilli

There are only a few on a cell and they are longer than fimbrae. Sex pilli are responsible for donor + recipient cells in bacterial conjugation

Type 4 pilli support adhesion and twitching mobility

What is the function of flagellum

Used for locomotion. Hollow and made out of the protein flagellin

What are the 4 types of flagellum and how do they differ

1. Monotrichous - A singular flagellum on one polar end of cell

2. Lophotrichous- Multiple flagella on one polar end of cell

3. Amptrichious - A singular flagellum on each polar end of cell

4. Petritrichous- Flagella surrounding the cell

Why is gene expression controlled in prokaryotes

Resource preservation- transcribing and translating all the time is resource intensive

Regulating bacterial behaviour- infection, number of flagella, behaviour, biofilm and spore formation

Response to the environment- Circadian rhythms, Quorum (being able to sense when other bacteria are around), immune evasion in pathogenic bacteria, gravitaxis

How are spores formed

What are sigma factors

• A subunit of RNAP holoenzyme

• Acts as a landing pad for the rest of RNAP

• Initiates binding and then falls off

• The binding is initiated in promotor sequences

What are 3 methods of controlling gene expression

1. Only activate some genes at once (use different promotor regions for different genes, requiring different sigma factors, however this requires a lot of different sigma factors)

2. Inhibit genes until we need them (use repressor/regulator genes which prevent sigma binding, or use anti sigma factors which sequester the sigma factor)

3. Degrade sigma factors (Use degraders to degrade the sigma factor before binding)

Example of controlling gene expression by degrading sigma factors

• Uses the gene RPOH and the sigma factor H

• Chaperone proteins (proteins which handle sigma factors) bind to the sigma factor and degrade it at 30 degrees, so RPOH isnt translated

• However if heat shock occurs some of the proteins denature and unfurl, causing the chaperone proteins to be sequestered to refold the proteins

• This means the sigma factors can activate RPOH

What are regulator proteins

Proteins which bind to the operator region

What is the operator region

Usually near the transcribed region

What is an operon

A cluster of genes in the same functioning unit

What is polycistronic

Described how multiple proteins can be produced from 1 mRNA

What is the function of operator regions

1. repressor protein binds to operator

2. Hinders DNAP (steric hinderance)

3. Genes are not transcribed

What are the 2 types of negative gene regulation

1. Repression - uses a repressor binding to operator region

2. Induction - inducer binds to repressor, removing it (eg LAC operon)

What is positive gene regulation

Induction - Inducer binds to activator protein, which binds to operator this changes the DNA structure to induce transcription

What is EMSA (gel shift assay)

Proteins (eg. sigma factors) in the sample will bind to DNA fragments, meaning the fragments will migrate slower in gel electrophoresis. The labelling of nucleotides enables the detection of the protein - nucleotide complexes

What are promotor and consensus sequences

Promotors are found at the 5 prime end and contain conserved consensus sequences which allow sigma factor binding. The consensus sequence is the most common order of nucleotides in a promotor sequence

How does the genome of prokaryotes and eukaryotes differ

Prokaryotes- Circular DNA, 1 chromosome, lacks introns

Eukaryotes - Linear DNA, multiple chromosomes in pairs, introns

How does the DNA location of prokaryotes and eukaryotes differ

Prokaryotes- Nucleoid region in cytoplasm

Eukaryotes- Within membrane enclosed nucleus

How does the cell wall of prokaryotes and eukaryotes differ

Prokaryotes- usually present and made of peptidoglycan

Eukaryotes- when present made of cellulose or chitin

How does the internal membrane of prokaryotes and eukaryotes differ

Prokaryotes- May have energy inducing lamella

Eukaryotes- Extensive membranous organelles

How does the gene structure of prokaryotes and eukaryotes differ

prokaryotes- Contains operator region and no introns/exons

Eukaryotes- No operator region but includes introns and exons

How is RNA processed in eukrayotes

• Introns are spliced out, exons are ligated

• Forms mRNA

• Called splicing

• Novel proteins can be created by ligating exons in different ways

What is compartmentalisation

It enables specialization of cells, allowing different biological processes to happen simultaneously and separately. Allows different reactions to have different conditions (eg. pH)

What is the golgi apparatus

Made out of membrane bound stacks of organelles called cisternae. Post transcriptional modifications occur here.

What is the endosomal system

Acts as a “way station” for proteins coming in/out of the cell

How does the nuclear pore complex control import/export

It is a multi unit holoenzyme, proteins and nucleic acids can pass if they have the NIS (proteins) or are attached to a chaperone protein (nucleic acids).

What is the NIS

Nuclear localisation signal, a sequence of amino acids which is required for proteins to enter the nuclear pore complex.

What is the difference between heterochromatin and euchromatin

Heterochromatin- tightly wound, inactive chromasomes tethered to the nucleus by lamina

Euchromatin- active DNA, less tightly wound

What does the mitochondrial genome contain

Circular DNA, genes for the ETC subunits and ribosome function (tRNA). Proteins are imported from nucleus by chaperone proteins.

How does the endomembrane system transport proteins

Endomembrane system consists of

• Endoplasmic reticulum

• Nuclear membrane

• Golgi apparatus

• Vesicles

• Lysosome

Proteins are shuttled from the ER to the golgi to a vesicle

What do the free ribosomes in cytosol produce

Proteins which are shuttled to the nucleus, chloroplasts, mitochondria and peroxisomes

What do the ER membrane bound ribosomes produce

Proteins which are shuttled to the golgi apparatus which are then transferred to secretory vesicles to the plasma/nuclear membrane, endosomes or lysosomes

What are the 3 types of immunostaining

1. Immunofluorescence- Uses florescent dye

2. Immunohistochemistry- Uses antibodies in a tissue

3. Immunocytochemistry- Uses antibodies in a cell

What are some advantages of multicellularity

• Cells are bigger and therefore have greater protection from predation and better buffering from environment

• Allows specialised cells to develop

Explain the flagellar synthesis constraint hypothesis

• Flagella allow a simple multicellular organism to move.

• However the microtubule organising machinery needed for flagella formation is also needed for spindle fibre apparatus in cell division

• Therefore there is competition for the microtubule machinery

• The presence of both flagellated and non-flagellated cells allows movement and development in one simple colony

Explain weissmans nuclear determinants theory

Unequal division of determinants leads to cell specialisation and differences.

Explain Hans Dreish’s experiments and theory

• He used sea urchin blastomeres (2 cell embryos) to observe development

• Saw that each blastomere developed into a complete larva

• Shows early embryonic cells retain ability to develop into a full organism

• Disproves weissmans nuclear determinants theory

Why is cell-cell signalling important

• Balance between differentiated cell types (flagellar constraint hypothesis)

• Homeostasis and maintenance of internal conditions

• Regulating development

• Recognising self and non self

• Cell adherance

What is cell lineage

The cells which are switched on/off in a cell. Important as all cells have the same genetic material so there must be a key process regulating gene action in development.

Eg. Dolly the sheep

What are the different types of cellular response

• Movement (eg. slime mould)

• Contraction (eg. lactation)

• Metabolism alteration (eg. Diabetes response)

What is gene expression

The process by which a genes coded information is converted into the structures which are present and operating in the cell

How is gene expression altered

• A gene may/may not be translated into RNA

• Splicing

• At export from nucleus

• Proteins encoded by genes may be regulated by post-transcriptional modifications that alter activity and stability of the protein

How is a eukaryotic protein coding gene organized

Enhancers/silencers - Appear throughout genome

Promotor- Composed of core and proximal regions

Open reading frame- Contains exons and introns

What is the difference between cis and trans regulatory elements

Cis - Regions of DNA involved in gene regulation (enhancers, silencers, promotors)

Trans- binding factors which bind to cis regulatory elements (eg. transcription factors)

How do transcription factors operate

They alter the activity of cis regulatory elements by binding to the major groove of DNA (does not melt it)

What are the functions of general transcription factors

They form the RNAP complex at the TATA box

What is the function of TBP (transcription binding protein)

1. Recognises TATA box

2. Bends the DNA 80 degrees to separate strands

3. As there are fewer H bonds between TA and GC, it is easier to melt at the TATA box

What is the full mechanism of RNAP complex binding in eukaryotes

1. TF11D (made up of TBP and TAF) recognises TATA box and separates strands

2. TF11B recognises binding recognition sequence in promotor region and positions RNAP at the start site

3. TF11F attracts and stabilises RNAP interaction with TF11H and E

4. TF11H and E unwinds at start point and phosphorylates. Releases RNAP from promotor

5. RNAP complex is formed, transcription factors are released and transcription begins.

How do specific transcription factors function

They recognise regions of 6-12 bases long (motifs) and influence the binding of transcription initiation complex by binding to silencer/enhancer regions. They can also recruit other proteins to these regions.

How do specific transcription factors influence silencers / enhancers

They form loops in the DNA to bring the regions back into proximity with the promotor

What is the wnt signalling pathway used for

Embryonic development and tissue regeneration

What is the process of the wnt signalling pathway

1. wnt messenger molecule activates the frazzled receptor in the cell membrane

2. This activates dishevelled which inhibits the b catenin destruction complex

3. This leads to the stabilisation of b catenin which activates transcription factors

However if the pathway is mutated, the transcription factors wont be activated as b catenin is still degraded.

How does the hypoxia pathway function in normal oxygen conditions

1. HIF1A is marked by proline hydroxylase which requires oxygen

2. Marked HIF1A is recognized by pVHL which targets it for degredation

3. No genes are activated

How does the hypoxia pathway function in hypoxic conditions

1. HIF1A cannot be marked due to lack of oxygen

2. It is not degraded so will bind to genes and activate them

3. Eg. VEGF which is the gene for blood vessel developement

How is binding of a transcription factor measured

CH1P - qPCR

1. DNA is crosslinked to transcription factors using formaldehyde

2. DNA is sheered using ultrasound and purified using antibodies for the transcription factor

3. The region in which TFs are bound is amplified

4. If no TFs are present then the region is not amplified.

What is extracellular signalling

The process by which cells communicate with each other via signalling molecules 

What is the function of receptors

Enable the cell to respond to a signal 

• The carriers of these signals will bind to specific receptor proteins. 

What are the 4 types of signalling

1. Contact 

   1. Signalling requires cells to be in direct contact between signaling and receiving molecules 

2. Paracrine

   1. Signalling secreted in the extracellular space

3. Synaptic 

   1. Specialized paracrine using neurotransmitters released in synapses between neurons 

4. Endocrine 

   1. (hormones) produced in a local group of cells and secreted 

What are some examples of signalling molecules

Peptides, e.g. Insulin/VEGF. These are the products of genes and encoded like any other protein.

Small molecules, e.g. nitrous oxide – short lived and breaks down fast.

Metabolic products, e.g. Steroids. These are not encoded in genes but are the product of a series of enzymes. 

Lipids, e.g. phospholipids. These remain bound to membranes.

What are the different types of signal termination

Receptor internalisation/sequestration

•  the receptor is brought into the cell so it can no longer respond to the signal. 
  

Degradation/Down-regulation of the signalling molecule/receptor:

•  The molecule may break down on its own or enzymes may break down the signalling molecule receptor. 

Feedback inhibition: 

• Activation of the receptor leads to feedback loops that make the receptor less responsive to activation.

How do hydrophilic molecules cross the plasma membrane

As the receptor is bound to the surface the transduction is usually more complex, as the signal has to pass through the cell surface and cytoplasmic and onto the intracellular target. These can be very complex. 

How do hydrophobic molecules cross the plasma membrane

They can cross the membrane so they use intracellular receptors

Often transported by carrier proteins, they can diffuse across the membrane; the receptor can be found in either the cytoplasm or even the nucleus. The consequence is the signal transduction pathway is less complex.

What are steroid hormones

• Synthesized from cholesterol

• Small organic molecules 

How do steroid hormone receptors operate

Nuclear Hormone Receptors are both receptors and transcription factors. This feature of the receptor means it directly connects the binding of the steroid as a ligand to the transcription of a gene.

In the absence of a hormone, the signaling molecule and ligand for the receptor. The receptor is held in an inactive complex. Typically this is a monomer in bound to heat shock proteins. 

Binding of the ligand to the receptor alters the shape of the nuclear receptor, activating it. The active form then releases from inhibitory proteins and acts as a transcription factor. 

What is amplification

Amplification in the pathway can be achieved by a single receptor activating an enzyme. 

The enzyme may then activate many other proteins or generate many other secondary messenger molecules. The result is that one molecule (an enzyme) amplifies the signal of many other molecules to enable the cellular effect. 

What is integration

Integration in a pathway is often caused by two signalling pathways by using a shared common component. 

This means two signals can activate the same downstream effect.  Alternatively, the second pathway may inhibit the output of the first to provide regulation of the second pathway. 

How does regulation of signalling pathways operate

A single pathway can be regulated at multiple points, using the Wnt pathway as an example again. 

Eg.

• The Frizzled receptor expression 

• Regulation of β-catenin Stability

• TCF/LEF Transcription Factors (and cofactors)

• Crosstalk with other pathways, e.g. Notch

What is protein phosphorylation

The addition, or removal, of a phosphate group to a protein can alter the structure and activity of the protein. The phosphate group is typically added to a tyrosine residue in the protein, but serine, threonine are also common targets for phosphorylation. The phosphate group is highly charged, and therefore alters the protein confirmation. One third of eukaryotic proteins are phosphorylated.

What are protein phosphatases and kinases

Kinases

• Specific to the residue of the phosphate 

• Specificity is determined by the amino acids which surround the target amino acid residue, these are recognized as the kinases 

Protein phosphatases 

• Remove phosphate groups from proteins, they catalyze the reverse reaction to remove the phosphate group. 

What is SDS page

SDS-PAGE stands for Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.

The technique separates proteins based on their molecular weight by using an electric field to move them through a polyacrylamide gel.

How are the proteins treated before being analysed using SDS page

Proteins are treated with SDS, a detergent that denatures them and imparts a uniform negative charge, and mercaptoethanol to remove disulphide bonds.

This denaturation ensures that proteins unfold into linear structures and carry a consistent negative charge per unit mass, facilitating separation based on size alone.

What is the process of western blotting used for

Western blotting allows for the specific detection of a target protein within a complex mixture. This uses specific antibodies. It also generates quantitive data by measuring the intensity of bands on the blot . This is useful for comparing protein expression between different samples or conditions.

What steps are involved in the process of western blotting

Gel Electrophoresis 

We initially run an SDS-PAGE gel, but don’t stain.

Transfer to Blotting Membrane 

After separation, the proteins are transferred onto a membrane. This transfer allows the proteins to be immobilised and accessible for antibody binding.

Incubation with Antibodies

The blotted membrane is then incubated with primary antibodies, which specifically recognise and bind to the target protein of interest. Following this, secondary antibodies that are conjugated to enzymes or fluorescent markers are introduced. 

Detection Methods 

Depending on the type of secondary antibody used, the detection of the target protein can occur through various methods.  

Why are secondary antibodies used

Amplification of Signal

Secondary antibodies amplify the signal generated by the primary antibody. This amplification is crucial for enhancing the detection sensitivity, as secondary antibodies can bind to multiple sites on the primary antibody, leading to an intensified signal.

 

Cost-Efficiency

Secondary antibodies are generally less expensive to produce than primary antibodies. Using a universal secondary antibody with various primary antibodies can be a cost-effective approach, especially when conducting experiments involving multiple target

Describe fluorescent detection as a secondary antibody detection method

Secondary antibodies are conjugated with fluorescent dyes.

Fluorescent signals are visualised using a fluorescence microscope or gel documentation system.

Pros- High sensitivity and multiplexing capabilities, minimal sample degradation

Cons - Potential for photobleaching

Describe chemiluminescent as a secondary antibody detection method

Enzyme-catalysed reaction produces light, often through luminol or other chemiluminescent substrates.

Emitted light is captured and visualized using specialized equipment.

Pros - High sensitivity and wide dynamic range, minimal background noise

Cons- Relatively short signal duration, limited options for multiplexing.

Describe colorimetric detection as a secondary antibody detection method

Enzymes (e.g., horseradish peroxidase) conjugated to secondary antibodies catalyse a reaction producing a visible product.

Colorimetric (colour change) or chemiluminescent (light emission) signals are generated.

Pros- Moderate sensitivity

Cons- Signal decay over time, limited dynamic range

Describe radioactive detection as a secondary antibody detection method

Utilizes radioactive isotopes or labelling probes.

Radioactive emissions are detected by autoradiography.

Pros - High sensitivity, quantitive measures

Cons- Safety concerns due to radioactivity, short half life of some isotopes.

Why are positive controls used in western blots

Importance: Confirming the efficacy of antibody binding, detection, and overall success of the western blot experiment.

Example: Known protein sample with expected band size under the chosen experimental conditions.

Why are loading markers/housekeeping proteins used in western blots

Importance: Normalizing for variations in sample loading, ensuring equal protein amounts, and validating consistent transfer efficiency.

Example: Housekeeping protein, like GAPDH, serving as an internal control for gel-based western blot analyses.

Why are negative controls used in western blots

Importance: Assessing antibody specificity, detecting potential contamination, non-specific binding and preventing false positives.

Example: Using a cell line that doesn’t express the protein. 

Why are molecular weight markers used in western blots

Importance: Facilitating accurate size estimation of target proteins, confirming successful transfer, and aiding in result interpretation.

Example: Confirming band is appropriate mass for the known protein, and therefore specific.

What are RTKs

Receptor tyrosine kinases

• They exist for many different signals and all function by a similar pathway for activation by dimerisation to a ligand.

  RTKs form the largest family of cell surface receptors and are directly the cell surface to an intracellular enzyme that can phosphorylate tyrosine.