Cambridge Biology of Cells Year 1 Michaelmas Term

What type of cells are commonly used as starting material in cultures?

Because they contain rapidly growing cells, embryos or tumors are frequently used as starting material.

Fluorescence and recombinant DNA?

DNA coding for GFP attached to a DNA sequence in the genome you are interested in, so when the protein you are interested in is made in the cell, GFP will be attached to it.

mRNA

However DNA does not direct protein synthesis itself but uses a closely related nucleotide polymer RNA (ribonucleic acid) as an intermediary

How do you access the information in DNA

DNA helix has major and minor grooves Information can also be read by "feeling" the structure of the bases exposed in the major groove

What is coding DNA?

DNA that encodes information to make proteins

Identify male pupae (drosophila)

Dark sex combs on front legs.

In order to preserve compartment size, what about the trafficking must be balanced?

Trafficking depends on shuttling vesicles between compartments and must be balanced by reverse traffic to preserve compartment size

Amino acid Phe?

Phenylalanine

Tell me the structure of phosphatidic acid

Phosphatidic acid consists of a glycerol backbone, with, in general, a saturated fatty acid bonded to carbon-1, an unsaturated fatty acid bonded to carbon-2, and a phosphate group bonded to carbon-3. "The difference from phospholipids is that their phosphate group has additional groups like choline etc " This is my impression, so double check

Draw a disulfide

R-S-S-R'

Tell me about plant cell vacuoles

Plant cell vacuoles have lysosomal function, can act as storage depots, control turgor pressure and influence cell growth

What is pectin

a highly hydrated polysaccharide found in the cell walls of plants pectin is what makes up the middle lamella which cements the cell walls of two adjoining plant cells together.

What is Bacteriorhodopsin

a light activated proton pump

Shine Dalgarno sequence can facilitate simultaneous ORF translation because....

Shine Dalgarno sequence can facilitate simultaneous ORF translation because ribosomes just need to scan for a shine dalgarno sequence and bind to one, then it can immediately begin transcription. Shine dalgarno sequence allows modular translation or ORFS, and hence is responsible for the capacity of prokaryotes to be polycistronic.

What factors help the ribosome bind to the AUG start codon.

Shine dalgarno binds to the 16s rRNA, this helps the 30S subunit(which is itself + some other proteins), bind to the AUG start codon. So the shine dalgarno binds to the 16s which helps itself bind to the AUG start codon. Initiation factor 3 also helps the ribosome bind to the start codon

Compound microscope: How wide should the condenser iris be opened?

The condenser iris diameter should be 2/3 of the original whole field(ignore the small black circle)

Which is bigger, chloroplasts or mitochondria?

chloroplasts are much larger

2 structures that can propel cells forward?

cilia - move fluid, flagella move cell

Tell me about bacteria that hijack actin filaments

an example is Listeria - hiijack actin filaments, actin polymerisation that propels bacteria through cytoplasm (up to 1µm sec-1) such as Shigella (dysentery) and Listeria, (can cause gut infections in newborns), to allow them to move around in the viscous cytoplasm of the eucaryotic cells they infect. The bacteria induce the assembly of actin filaments at one end. The growing filaments push the bacterium through the cytoplasm at rates of up to 1µm/sec, producing a rocket like tail, which disassembles as the bacterium moves forward and the tip meets disassembling factors in the cytoplasm.

Define centrosome

an organising centre for microtubules that acts as the spindle pole during mitosis

Tell me about anabaena

anabaena photosynthetic bacterium

tRNA - transfer RNA

decodes mRNA, allowing protein sequence to manifest. adapter molecule

Glossary: Plastid

double membrane-bounded organelle in plant cells, including chloroplasts

glossary: eukaryotic

eucaryotic: pertaining to cells with a nucleus and complex cytoplasmic organelles (cf. procaryotic)

What are protozoa

free living eukaryotic cell

Glossary: Nucleolus

granular structure in the nucleus, associated with specific chromosomes, involved in ribosome synthesis and assembly

Glossary: Pilus (pl pili)

hair-like structures on procaryotic organisms, involved in bacterial recognition and conjugation

Tell me about reformation of the nuclear envelope near the end of mitosis

reformation of nuclear envelopes, the incorporation of nuclear pore complexes and the re-assembly of the nuclear lamina.

Glossary: Peroxisome

single membrane bound organelle involved in reactions producing or consuming hydrogen peroxide

Tell me 4 functions of the SER

smooth endoplasmic reticulum, unlike its "rough" counterpart, does not package protein. Instead it carries out various metabolic reactions within the confines of its membrane. Functions range from carbohydrate metabolism, lipid synthesis(synthesising steroid hormones), and even drug detoxification. Drug detoxification is usually accomplished by adding hydroxyl groups to drug molecules. This makes them more soluble in water and therefore easier to be flushed from the body. Another important function of the smooth endoplasmic reticulum is the storage of calcium ions. This is imperative for muscle contraction. When a muscle cell is stimulated by a nerve impulse, the calcium ions rush into the cytosol and trigger muscle contraction. Structurally the smooth ER is not continuous with the cell's nuclear envelope. The smooth ER is involved in phospholipid synthesis.

What is catabolic what is anabolic?

metabolic reactions leading to degradation metabolic reactions involved in synthesis

are viruses prokaryotic or eukaryotic?

neither Since viruses are acellular- they contain no cellular organelles, cannot grow and divide, and carry out no independent metabolism - they are considered neither prokaryotic nor eukaryotic.

Glossary: procaryotic

pertaining to a cellular organism lacking a membrane-bounded nucleus, eg mycoplasma, bacteria, (cf. eucaryotic)

where are the phosphoanhydride bonds in ATP?

phosphoanhydride bonds are high energy bonds

Define a cell

the smallest membrane-bounded unit capable of independent reproduction

What is the cytosol?

the soluble phase of the cytoplasm

Glossary: Periplasmic space

the space between the inner and outer membranes of Gram negative bacteria

What does "endoplasmic" mean? What does reticulum mean?

"within the cytoplasm" "Little net"

3 Ways to regulate bacteria gene expression:

1) Modulate DNA binding specificity by sigma factors. By changing the availability of sigma factors, you can change the genes that are regulated. 2) Protein repressors and proteins activators. Exemplified by the lac operon. 3) Attenuation

What is the sodium ion concentration in the blood plasma? What is the sodium ion concentration in cells?

135-145 millimoles/litre same as milliequivalents per litre in blood plasma about 10 mMol in cells

What is the nature of the binding of the shine dalgarno sequence with the 16s rRNA?

16s rRNA itself is also a nucleotide sequence. This means they can just pair by complementary base pairing.

In what direction is DNA read?

5' to 3'

What are dichroic mirrors?

(of a crystal) showing different colours when viewed from different directions, or (more generally) having different absorption coefficients for light polarized in different directions.

What proportion of the total membrane does the ER constitute in a cell?

1/2 on average

How many genes do most bacteria and archaea have?

1000-6000 genes

Tell me about Actin's interaction with myosin in chara cells

2) Actin's interaction with myosin promotes movement within cells example: large algal cells Chara corallina, Cytoplasmic streaming is very rapid. Practical class average was 40µm sec-1

How many chromosomes in humans?

22 PAIRS of autosomes, + 2 sex chromosomes genetic material of eukaryotes is divided into chromosomes and packed into chromatin

How do small RNAs regulate gene expression?

3 types of small RNAs: 1) microRNA 2) small interfering RNA 3) short hairpin RNA

How many milliosmoles is 150 milliMolar of NaCl

300 milliosmoles, because NaCl splits up

Lipid bilayer

5nm bilayer is amphipathic maintain integrity of the cell selectively permeable

What provides the motive force for chromosome separation?

A complex combination of motor protein activity and regulated microtubule disassembly provides the motive force for chromosome separation.

What is a diacylglycerol?

A diglyceride, or diacylglycerol (DAG), is a glyceride consisting of two fatty acid chains covalently bonded to a glycerol molecule through ester linkages.

How did plant cell walls originate?

A eukaryotic cell equipped with chloroplasts has no need to chase after other cells as prey; it is nourished by the captive chloroplasts it has inherited from its ancestors. Correspondingly, plant cells, although they possess the cytoskeletal equipment for movement, have lost the ability to change shape rapidly and to engulf other cells by phagocytosis. Instead, they create around themselves a tough, protective cell wall. If the first eukaryotic cells were predators on other organisms, we can view plant cells as cells that have made the transition from hunting to farming.

Where does ATP generation occur on prokaryotes?

ATP generation by chemiosmotic coupling occurs on the plasma membrane of procaryotic cells

What is Numerical aperture?

A measure of the range of angles from which a lens can accept light. A measure of the amount of diffracted light that the lens can accept.

ATP is coupled to what?

ATP often coupled to other reactions, eg synthesize a peptide bond

Tell me about indirect immunofluorescence

A way of amplifying the signal. It requires two incubations with different antibodies. The primary antibody binds specifically to the macromolecules of interest. The primary antibody is then visualised by incubating with a secondary antibody (specific for the first antibody), which has been covalently linked to a fluorescent dye. Brandon: How does it Amplify? My explanation:(only one antibody can bind to the original protein, but two fluorescent antibodies can bind to the first antibody, hence amplifying the signal) this is my hypothesis not confirmed

What are organotrophs?

An organotroph is an organism that obtains hydrogen or electrons from organic substrates. Organotrophic species can utilize virtually any type of organic molecule as food, from sugars and amino acids to hydrocarbons and methane gas.

Tell me about the model plant chosen by the scientific community

Arabidopsis Has Been Chosen Out of 300,000 Species As a Model Plant It is a small weed can be grown indoors in large numbers and produces thousands of o spring per plant after 8-10 weeks. Arabidopsis has a total genome size of approximately 220 million nucleotide pairs, about 17 times the size of yeast's

Define cisterna

Membrane-bound compartment, usually flattened, as in the Golgi body or endoplasmic reticulum

What are microsomes?

Broken membranes which have resealed into small closed vesicles a vesicular membrane preparation of the endoplasmic reticulum produced by homogenization and centrifugation

Tell me about C. elegans

Caenorhabditis elegans (Figure 1-39) is a small, harmless relative of the eel- worm that attacks crops. With a life cycle of only a few days, an ability to survive in a freezer inde nitely in a state of suspended animation, a simple body plan, and an unusual life cycle that is well suited for genetic studies C. elegans develops with clockwork precision from a fertilized egg cell into an adult worm with exactly 959 body cells (plus a variable number of egg and sperm cells)—an unusual degree of regularity for an animal.

Tell me the two common fixatives used in electron microscopy and what do they do?

Cells are usually fixed using glutaraldehyde which covalently cross-links protein molecules to their neighbors and then with osmium tetroxide which binds to and stabilizes lipid bilayers as well as proteins. Brandon: Cross link How? Does Aldehyde react with amine from protein forming imine??? "Becomes reduced" due to strong reducing environment in the cytosol

What does glycogen phosphorylase do?

Cleaves glucose of glycogen one at a time, releasing glucose-1-phosphate

Give some examples of restriction enzymes

EcoRI HindIII BamHI

Tell me the general differences between eukaryotes and prokaryotes in terms of organelles, chloroplasts, mitotic spindle, lysosomes, etc present in the cytoplasm.

Eukaryotes Internal membrane-bound organelles such as: Mitochondria Endoplasmic Reticulum Golgi apparatus vacuoles lysosomes are present. c. Chloroplasts serve as organelles for photosynthesis. d. A mitotic spindle involved in mitosis is present during cell division. prokaryotes b. Internal membrane-bound organelles such as: mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and lysosomes are absent c. There are no chloroplasts. Photosynthesis usually takes place in infoldings or extensions derived from the cytoplasmic membrane. d. There is no mitosis and no mitotic spindle.

Give me some numbers for diversity of size and shape of cells

Eukaryotic cells can be 100µm, most are 30-50 Yeast cells - single celled eukaryotes ~5µm

What role does filter paper have during drosophila testes dissection?

Filter paper slowly removes liquid between coverslip and slide, which gently squashes the sample.

Tell me about FRAP experiment

Fluorescence recovery after photobleaching (FRAP) is a method for determining the kinetics of diffusion through tissue or cells. It is capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells. This technique is very useful in biological studies of cell membrane diffusion and protein binding.

Tell me about the formation of NADH during the formation of acetyl CoA via the pyruvate dehydrogenase complex

Formation of NADH: The final step in this reaction occurs when the oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3).Two electrons are transferred to first an FAD prosthetic group of the enzyme and then to NAD+. This process of transferring electron is very unusual because FAD are known to receive electrons from NADH, not transfer them. Within the enzyme, the electron-transfer potential of FAD is increased by its chemical environment which enables it to transfer electrons to NAD+. Flavoproteins are proteins which are tightly associated with FAD or FMN also known as flavin mononucleotide. Dihydrolipoamide + FAD --> Lipoamide + FADH2 + NAD+ --> FAD + NADH + H+

What is the purpose of gluconeogenesis

Gluconeogenesis is a process, in which Pyruvate (a product of Glycolysis) is backward-converted into sugar, glucose in particular. Which can then be stored in the form of glycogen in animals' cells or starch and cellulose in plants' cells.

How can chromatin regulate gene expression?

Histones - methylation, acetylation - lysine DNA - methylation Heterochromatin/Euchromatin Key enzymes involved in regulating gene expression via chromatin: histone acetylases and deacetylases Epigenetics: 3 different types: 1) Cellular memory/control induced by tissue-specific transcription factors. Involved in development. 2) transcriptional - environmentally induced. Primes cells to respond more quickly to a repeat challenge. 3) transgenerational - cellular stresses that change the chromatin of gametes. Can be retained for a few generations.

How does centrosome duplicate?

In the cytoplasm the centrosome duplicates through the separation of the two centrioles, each of which initiates the assembly of a new single centriole, to produce two paired structures.

What happens if you leave testes out for too long?

If you leave them out for more than 5 minutes they begin to disintegrate

List the 5 animal cell models most commonly used in research in the scientific community

In order of increasing size, they are the nematode worm Caenorhabditis elegans, the y Drosophila melanogaster, the zebra sh Danio rerio, the mouse Mus musculus, and the human, Homo sapiens. Each has had its genome sequenced.

Role of microtubules in plants?

In plants microtubules play an important role in determining cell shape by organising the orientation of cellulose microfibrils deposited in the growing cell wall. There is a coincidence between the regular orientation of microtubules just below the plasma membrane and the alignment of deposited fibrils in the cell wall.

Using infrared light vs visible light in microscopy?

Infrared laser light causes less damage to living cells than visible light and can also penetrate further, allowing microscopists to peer deeper into living tissues.

What is the main objective a scientist wants to achieve when they perform subcellular fractionation?

Isolate organelles of eukaryotic cells, so can be studied/harvested. Usually accomplished by differential centrifugation.

Tell me about the reproduction of S. cerevisiae

It can reproduce either vegetatively (that is, by simple cell division), or sexually: two yeast cells that are haploid (possessing a single copy of the genome) can fuse to create a cell that is diploid (containing a double genome); and the diploid cell can undergo meiosis (a reduction division) to produce cells that are once again haploid In contrast with higher plants and animals, the yeast can divide indefinitely in either the haploid or the diploid state, and the process leading from one state to the other can be induced at will by changing the growth conditions.

What do kinesins and dyneins do? Which directions do kinesin and dynein move?

Kinesin and dynein are molecular motors which track along microtubules Kinesins and dyneins can transport material great distances e.g along microtubule tracks in the axon of a nerve cell kinesins, which track towards the plus end and dyneins, which move towards the minus end

4 main classes of junctions in eukaryotic cells?

Junctions between eucaryotic cells can be categorized into 4 main classes: anchoring or adhesive junctions, occluding (or tight) junctions, channel-forming junctions and signal-relaying junctions. We shall focus on adhesive and channelforming junctions

What protein regulates the entry and exit of mRNA via the nuclear pore from the nucleus into the cytoplasm

Nuclear export receptors

Tell me about adhesive junctions

Of all cell interactions in multicellular organisms the most fundamental are those that hold cells together, either through adhesion to each other or to a common extracellular material that they secrete. In both types of adhesion the transmembrane junctional protein links the cytoskeleton inside the cell either to components of the extracellular material or to the partner junctional protein in the neighbouring cell (Figure 39). Actin and intermediate filaments are associated with different adhesive junction types. As well as stabilising close contacts between cells, adhesive junctions are often also sites of active signalling through extracellular signals and membrane receptor proteins. Adhesive junctions stick cells together and also link their cytoskeletal filaments into intercellular networks

What is the Pentose Phosphate Pathway important for?

Pentose Phosphate Pathway makes a precursor important for RNA/DNA synthesis. PPP generates NADPH and pentoses (5-carbon sugars) as well as ribose 5-phosphate (the precursor to creating DNA + RNA) The Pentose Phosphate pathway produces to important precursors for basic building blocks of life: The production of ribose for the synthesis of some cofactors, DNA, RNA. The production of NADPH for the synthesis of fatty acids, steroids, and other oxidation-reduction reactions (some occur in photosynthesis, especially in the Calvin cycle)

Tell me about porphyrins

Porphyrins are a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges (=CH−). Picture shows Structure of porphin, the simplest porphyrin

How do cells direct proteins to the target site? Outline the sequence of events

Protein fate is determined by a signal sequence. Signal sequence is recognised. Receptors ensures events are localised to the ER

Glossary ribosome

RNA-protein structure, the site of protein synthesis in eucaryotic and procaryotic cells. Also present in mitochondria and plastid

Prepare pancreatic acinar cells?

Remove paraffin wax with histoclear Add ethanol (removes histoclear) Distilled water(removes ethanol) stain with pyronin and methyl green Rinse with water to remove excess stain Blot with filter paper to remove excess water. Dry Add one drop of mounting medium over section. Hot plate Light micreoscopy

Tell me the 3 catalytic cofactors involved in the Pyruvate Dehydrogenase Complex

The catalytic cofactor includes coenzymes such as thiamine pyrophosphate (TPP), lipoic acid, and FAD.

What is a fluorescent dye? Concentrations of fluorescent molecules?

The fluorescent dye is a molecule that absorbs light at one wavelength and emits light at a longer wavelength. Fluorescent molecules are detectable at very low concentrations.

What is the genome

The full complement of hereditary information in an organism

How does numerical aperture affect resolving power?

The greater the resolving power, the smaller the minimum distance between two lines or points that can still be distinguished. The larger the N.A., the higher the resolving power.

What are the two requirements for information storage for biological systems?

The information storage system must be: 1) Stable but accessible. 2) Able to be easily replicated without introduction of errors.

What is the spatial resolving power?

The minimum distance at which two objects can be distinguished.

What are cilia and flagella made of?

These are motile structures built from microtubules and dynein. They appear as hair-like appendages, about 0.25 µm in diameter, projecting from the cell surface.

Tell me about the permeability of the membrane in chloroplasts

They have a highly permeable outer membrane and a much less permeable inner membrane.

Why must samples viewed with light microscopy be dehydrated?

To cut into thin sections, cell needs to be embedded in paraffin wax Wax not soluble in water

Tell me about the contractile ring in cytokinesis

To form two cells the cytoplasm must split and here it is the actin cytoskeleton that is active in animal cells. Actin forms a contractile ring at the level of the spindle equator and through the motor activity of myosin, pulls the cell membrane inwards to form a deepening furrow, which eventually pinches off to produce two daughters. Contractile ring plays a role in the splitting of the cell to form 2 daughter cells. pinching of the cytoplasm around the contractile ring during cytokinesis

Steps to perform immunofluorescence?

Uses antibodies (isolated from the blood of an animal which has been previously injected with an antigen) that bind to a particular protein you are interested in. Cells are first fixed, the cell membrane permeabilised with a detergent that opens holes in the membrane to allow the large antibody molecules to enter the cytosol. The cells are incubated with the primary antibody which has been covalently linked to a fluorescent dye. After washing away the unbound antibody, the bound antibody is visualised by fluorescence microscopy

In TEM, what is the wavelength of the electron dependent on? What resolutions can TEM achieve, and what magnifications? Resolution of SEM?

Wavelength is dependent on the speed of the electron beam, which itself is dependent on the accelerating voltage. Electron wavelength affects resolution. TEM resolutions can be about 1.0 nm or better, magnifications can be about 10 000 000x. It has been possible to resolve atoms in silicon with a resolution of 78pm, and a magnification of 50 000 000x. Typical resolution of SEM is about 10 nm.

How do you work out the structure of ribosomes?

X-ray crystallography

Tell me about fluorescent dyes and ions Give an example of a dye that can image changes in Ca²⁺ concentrations

You can introduce fluorescent dyes that bind to specific ions, allowing the localisation and concentration of the ion in the cytosol to be determined. the dye fura-2 can be used to image changes in Ca²⁺ concentrations in cells

If you have some DNA, how do you visualise it

You can stain it with ethidium bromide and using UV light

Define chromatin

a complex of DNA and proteins, the nucleoprotein material of chromosomes

Tell me about differential centrifugation

a technique used to separate the components of cells on the basis of their size and density.

Tell me about archaea Differences between archaea and bacteria?

archaea are mostly extremophiles They have membranes composed of glycerol-ether lipids, whereas bacteria and eukaryotes have membranes composed mainly of glycerol-ester lipids. Ether bonds are chemically more resistant than ester bonds. This stability might help archaea to survive extreme temperatures and very acidic or alkaline environments. The stereochemistry of the archaeal glycerol moiety is the mirror image of that found in other organisms.

Glossary: Nucleosome

bead-like structure of DNA wrapped round an octamer of histones, the repeating subunit of chromatin structure

What do you mean by fixation?

biological tissues are preserved from decay by terminating any biochemical reactions. Fixation increases the mechanical strength and stability of the tissues. In electron microscopy, fixation is necessary as tissues are exposed to a very high vacuum, thus fixation is required to keep these cells in tact.

Words: cell division and cell cycle

cell division is ONE PART of the cell cycle

Compound microscope: How do you centre the condenser?

centring pins

How to increase efficiency of metabolism

compartmentalisation - allows the local concentration of reactants to be changed therefore increasing efficiency of metabolic reactions (especially in eukaryotes) proteins can be organised into arrays in membranes to maximise efficiency eg patch of bacteriorhodopsin in bacteria

Tell me about x-ray crystallography

diffraction pattern produced from beaming x-rays through the protein crystal. diffraction pattern gives information about the location of atoms(electron density) in the crystal. Computers use the diffraction pattern to reconstruct the protein lattice. X-ray crystallography allows us to work out protein structure, which allows us to work out how the protein functions. B: Can x-ray break bonds in protein?

condensation reactions

endergonic

Compound microscope: How do you check that the condenser is in focus?

i dont know

crista (pl. cristae)

infolding of the inner membrane of the mitochondria

30S subunit is made of?

its made of the 16s rRNA and multiple prokaryotic proteins.

Tell me about lysosomes

lysosomes are an acidic compartment rich in digestive enzymes

Compound microscope: How do you alter the diameter of the condenser iris?

move the condenser iris lever

histone

octamer

RNA to protein

one way street, compared to rna and dna which can go both ways

Compound microscope: How wide should the field iris be opened?

open field iris until its edge just disappears. This will ensure the whole field of view is illuminated with bright and even light, but regions outside of the field of view are not illuminated.

What does buccal mean?

relating to the cheek or mouth

ribosomes

ribosomes contain structures RNAs(rRNA) and more than 50 different ribosomal proteins

Essay Q: How is DNA's structure adapted to its function as an information carrying molecule?

tbc

Define centromere

the point at which a chromosome is attached to the spindle apparatus during cell division

2 mains ways to produce ATP?

use energy from sunlight or food

Hydrophobic and aliphatic side chains?

valine isoleucine leucine alanine

endosomes

vesicles that contain different enzymes

Tell me about the light source and power supply of the Zeiss Microscope

Ensure the brightness control is turned to its lowest setting (fully anticlockwise) and then switch on the microscope. You can now control the level of light using the brightness control. When you are not using your microscope, turn the brightness control to zero and then switch off the lamp. Always check that the brightness control is at its lowest setting before turning the microscope on or off, this extends lamp life.

What is contained in media used for animal cell culture?

In addition to salts and glucose, the media used for animal cell cultures contain various amino acids and vitamins, which the cells cannot make for themselves. The growth media for most animal cells in culture also include serum, which serves as a source of polypeptide growth factors that are required to stimulate cell division. Several such growth factors have been identified. They serve as critical regulators of cell growth and differentiation in multicellular organisms, providing signals by which different cells communicate with each other. For example, an important function of skin fibroblasts in the intact animal is to proliferate when needed to repair damage resulting from a cut or wound. Their division is triggered by a growth factor released from platelets during blood clotting, thereby stimulating proliferation of fibroblasts in the neighborhood of the damaged tissue. The identification of individual growth factors has made possible the culture of a variety of cells in serum-free media (media in which serum has been replaced by the specific growth factors required for proliferation of the cells in question)

In anabolic reactions, electrons are ______ to the molecule In breaking down molecules, electrons are _______ from the molecule A) Donated B) Removed Can be used more than once

In anabolic reactions, electrons are donated to the molecule In breaking down molecules, electrons are removed from the molecule

How does cell division occur in plants?

In plants the cell divides by the formation of a cell plate, again at the site of the spindle equator. Vesicles from the Golgi, filled with cell wall material, are directed by microtubules and fuse to form the membrane and cell wall between the daughter cells As we have seen, in prokaryotic cells tubulin-like filaments form a Z-ring at the point of cell division. Interestingly in dividing chloroplasts a similar protein performs an equivalent function.

What is a resin?

In polymer chemistry and materials science, resin is a "solid or highly viscous substance" of plant or synthetic origin that is typically convertible into polymers

Z-ring in prokaryotes?

In procaryotes tubulin-like filaments form the Z-ring at the site of cell division

Tell me about the actual formation of acetyl CoA via the Pyruvate Dehydrogenase Complex

In this step, acetyl CoA is formed when acetyl group is transferred from acetyllipoamide. This reaction is catalyzed by dihydrolipoyl transacetylase (E2). As the acetyl group is transferred to the CoA, the energy-rich thioester bond is preserved. Thus, the fuel for the citric acid cycle, acetyl CoA has been generated from pyruvate for use. Until the dihydrolipoamide is oxidized to lipoamide, the pyruvate dehydrogenase complex cannot complete another catalytic cycle. CoA + Acetyllipoamide --> Acetyl CoA + Dihydrolipoamide

Tell me about intermediate filaments

Intermediate filaments - form a meshwork They can be cross-linked into strong arrays to provide mechanical stability Intermediate filaments are 10-12nm in diameter and, unlike microtubules and actin filaments, are made up of elongated rather than globular subunits into filaments that have no polarity. They do not interact with motor proteins but can be cross-linked and bundled into strong arrays that give cells mechanical stability. One intermediate filament family are the keratins, which are restricted to vertebrate, nematode and molluscan cells. They stabilise the outer layers of the skin and are the structural basis of hair, nails and wool. Another family, nuclear lamins form a meshwork just inside the nuclear envelope, the nuclear lamina, which provides anchorage sites for chromosomes and nuclear pore complexes.

Tell me about Intermediate Filaments

Intermediate filaments are polymers of keratin. They help with cell shape also because it bears tension in the cell. It is primarily involved in organelle anchorage. The intermediate filaments also consist of various fibrous proteins that have a diameter of about 10 nm. Intermediate filaments often form a meshwork under the cell membrane and, in cells that lack a cell wall, help impart and maintain cell shape. Intermediate filaments are fairly stable and are not thought to undergo acute changes in length the way microfilaments and microtubules do.

What do intermediate filaments NOT do that microfilaments and microtubules do?

Intermediate filaments do not interact with motor proteins

Tell me about ion channels What properties of ion channels allow some ions to pass through, but not others? Tell me about some diseases associated with ion channels

Ion channels are proteins that forms water-filled pores through membranes, which allow specific ions to passively transport down the electrochemical gradient. For instance, the ion channels commonly found throughout the human body include Na+ channels, K+ channels, Ca2+ channels, and Cl- channels. These passive transport ion channels make use of the electrochemical potential to drive physiological processes, such as nerve impulse. The electrochemical potential is maintained by various active transport mechanisms, such as the Na+-K+ ATPase. The selectivity of the ion channels arise from the amino acids formation from which the protein is formed. Two main criteria determine what ions pass through these channels unimpeded: diameter of the pore and the electrical properties of the amino acids. The ion channels essentially forms a hole through the lipid bilayer of the membrane. The diameter of the hole helps determine which substances are allowed to pass through the channel. Only ions of an appropriate size will be able to pass through the hole of a specific ion channel. The observation that some larger ion channels only permit a specific ion to pass while smaller ions do not is based on the energetics of dehydrating the ion as it passes. The arrangement of amino acid carbonyl groups inside channels dictate the passage: the arrangement of the carbonyl groups is so that the dehydration energy costs are lowest for certain hydrated ions. Thus a larger pore has a favorable arrangement for larger hydrated ions, whereas a smaller hydrated ion passing through would require more energy to dehydrate. In potassium channels, this dehydration region that mediates the passage of K+ ions is known as the selectivity filter. In addition to the diameter of the pore, the electrical properties of the amino acid help determine which ions are allowed to pass through the ion channel. If the amino acids facing the inside of the pore are negatively charged, then only cations can pass through the channel. Anions will be repelled away from the negatively charged amino acids in the channel due to charge repulsion. Conversely, anions will be able to pass through channels lined with positively charged amino acids. There are some inherited ion-channel diseases. Some include: -Chloride-channel diseases include cystic fibroses and inherited tendency to kidney stones. -Potassium-channel diseases include some inherited life-threatening defects in the heartbeat, a rare inherited tendency to epileptic seizures in the newborn and several types of inherited deafness. -Sodium-channel diseases include inherited tendency to certain types of muscle spasms and Liddle's syndrome.

Tell me about ionophores Tell me about the potential therapeutic uses of ionophores

Ionophores are molecules that help transport ions from a hydrophilic environment into a hydrophobic environment. In other words, ionophores are ion carriers that help transport hydrophilic ions across lipid bilayer membranes. There are two broad mechanisms by which ionophores transport ions across cell membranes: carrier and channel forming. In the carrier mechanism, the ionophore forms a complex with the ion. The ionophore wraps around the ion with its polar interior. The exterior of the ionophore-ion complex is primarily hydrophobic, thus allowing the complex to cross the hydrophobic cell membrane. The carrier ionophore basically shields the ion's charge from the environment by solvation. For the channel forming mechanism, the ionophore induces a hydrophilic channel through the lipid bilayer membrane. The formation of this polar pore allows ions to cross through the cell membrane. Ionophores can be used as antibiotics due to its ability to disrupt the transmembrane electrochemical gradient. This electrochemical gradient is essential in driving metabolic processes. Without the gradient, there would be no net movement of ions into and out of the cell, essentially disrupting normal cellular processes. Ionophores disrupt the electrochemical gradient by allowing ions to freely diffuse across the cell membrane, either by forming complexes or channels. This free diffusion disrupts the normal ion balance between cytoplasm and the extracellular environment, thus eliminating the electrochemical gradient.

What is the relationship between distance migrated by a DNA/RNA/protein fragment against molecular weight?

It is useful to know that a plot of distance migrated by a fragment against log (molecular weight) gives a straight line relationship, meaning that running fragments of unknown sizes against known fragments can allow for an estimation of molecular weight.

Why is translation and transcription coupled in bacteria but not eukaryotes?

Its because of compartmentalization. Ribosomes do not exist in the nucleus, so translation cannot be coupled to transcription.

How does the lac operon work?

Key players of the lac operon: 1) cAMP 2) Glucose 3) Lactose 4) CAP 5) LacI cAMP activates CAP which binds to DNA and increases transcription of the lac operon by recruiting RNA polymerase. cAMP levels are inversely related to glucose levels. increase in lactose concentration increases concentration of allolactose which binds to LacI. LacI no longer represses the lac operon. Lac operon produces polycistronic mRNA.

What are lithotropic species Give an example

Lithotrophic species can feed on a plain diet of inorganic nutrients, getting their carbon from CO2, and relying on H2S to fuel their energy needs (Figure 1-16)—or on H2, or Fe2+, or elemental sulfur, or any of a host of other chemicals that occur in the environment. Beggiatoa, which lives in sulfurous environments, gets its energy by oxidizing H2S and can fix carbon even in the dark. Note the yellow deposits of sulfur inside the cells. (Courtesy of Ralph W. Wolfe.)

Why should you not use bright light to observe specimens under the microscope?

Living material under the microscope may be damaged by the intensity and heat of illumination: to minimise this (and to protect your eyes) don't use light brighter than that necessary for clear vision.

Tell me about what occurs in the nucleolus

Location where rRNA is synthesized and combined with proteins to assemble each ribosomal subunit. Subunits are exported and undergo final assembly in the cytoplasm.

Tell me about the lysosome How do lysosomes prevent self digestion?

Lysosomes are spherical bodies, or vacuoles that are enclosed by a single membrane. The membrane serves as a protectorate to the cell, since lysosomes contain harsh digestive enzymes, which would cause significant damage if exposed to cell content. Lysosomes contain different hydrolytic enzymes, such as proteases, lipases, and nucleases that are capable of breaking down all types of biological polymers (e.g. proteins, nucleic acids, carbohydrates, and lipids) that enter the cell or are no longer useful to the cell. In all, lysosomes function as the digestive system of the cell. On another note, lysosomes avoid self-digestion by glycosylation of inner membrane proteins, which prevent their degradation. Structure of Lysosome Lysosomes are membrane-enclosed organelles that help eukaryotic cells obtain nourishment from macromolecular nutrients. The lysosomes contain many hydrolytic enzymes such as proteases, nucleases, and lipases). The lysosomes are formed vesicles containing hydrolytic enzymes and proton pumps bud off from the Golgi complex. Phagocytosis and lysosomal digestion help the eukaryotic cell because they effectively increase the membrane surface area over which nutrients can be absorbed. However, in eukaryotes, lysosomes allow for intracellular digestion, and digested material crosses the lysosomal membrane into the cytoplasm.

Tell me about Metal shadowing in electron microscopy

Metal shadowing is another technique used to visualize the surface of isolated subcellular structures or macromolecules in the transmission electron microscope (Figure 1.34). The specimen is coated with a thin layer of evaporated metal, such as platinum. The metal is sprayed onto the specimen from an angle so that surfaces of the specimen that face the source of evaporated metal molecules are coated more heavily than others. This differential coating creates a shadow effect, giving the specimen a three-dimensional appearance in electron micrographs.

Tell me the composition and function of microfilaments. Tell me about the 3 types of cell shape.

Microfilaments are polymers of actin. They help with cell shape also because it bears tension in the cell. It is also involved in cell motility. There are three types of cell shape, which are microvilli, lamellipodia, and filopodia. Microvilli are projections on surface that increase surface area. Lamellipodia are membrane ruffles that help sense the environment and direct movement. Filopodia are like microvilli but are less stable. They also sense the environment. They can turn into lamellipodia. Microfilaments are formed when individual actin monomers polymerize, in a process fueled by ATP hydrolysis, to form chains of filamentous actin. Microfilaments are dynamic structures, growing and shrinking in a controlled manner. Some microfilaments play a structrual role in the cell to maintain cell shape. These structural microfilaments have protein caps at both ends to prevent changes in microfilament length. Anyways, other microfilaments have functions that require dynamic changes in length. The microfilaments also mediate cytoplasmic streaming, a mixing of the cytoplasm that aids diffusion.

Tell me about microtubule motors and their cargo

Microtubule motors can move cargoes of vesicles, greatly enhancing the efficiency of vesicle targeting and protein delivery. They may also move mRNAs to specific subcellular regions, allowing localised protein synthesis. They can move membrane-bound organelles or hold them in position. The ER has receptors that bind kinesin-like proteins on its surface and is spread out along the microtubules radiating out from the centrosome (plus ends). In contrast the Golgi body binds to dynein-like proteins and so tends to lie centrally in the cell, close to the centrosome (minus ends) and the nucleus.

Why can the assembly and disassembly of filaments (of the cytoskeleton) occur very quickly?

Microtubule motors can move cargoes of vesicles, greatly enhancing the efficiency of vesicle targeting and protein delivery. They may also move mRNAs to specific subcellular regions, allowing localised protein synthesis. They can move membrane-bound organelles or hold them in position. The ER has receptors that bind kinesin-like proteins on its surface and is spread out along the microtubules radiating out from the centrosome (plus ends). In contrast the Golgi body binds to dynein-like proteins and so tends to lie centrally in the cell, close to the centrosome (minus ends) and the nucleus.

Tell me about the composition and function of the Microtubules of the cytoskeleton Tell me about the two motor proteins that move along the microtubules

Microtubules are polymers of tubulin. They help with cell transport. They also help with the cell shape because it resists compression. It also helps facilitate cell motility. There are two motor proteins that assist organelles to move along the microtubules: Kinesin, which moves things away from the nucleus. Dynein, which moves things towards the nucleus. Microtubles have a larger diameter than microfilaments and intermediate filaments. The hollow microtubule structure consists of 13 tubulin dimers. They are one alpha-tubulin protein plus one beta-tubulin protein form one tubulin dimer. Microtubules also help movement of substances within the cell and are also involved in powering whole-cell movement by cilia and flagella. The microtubules provide tracks that can move vesicles from one organelle to the next in an efficient, directed fashion. Microtubules also segregate the duplicated chromosomes during mitosis.

tell me about genome of mito and chloroplasts

Mitochondria and Chloroplasts contain their own DNA and have 70S ribosomes, which support protein synthesis But only a small fraction of the proteins in these organelles are synthesized from their own genomes

Where are mitochondria found more often?

Mitochondria are found where the energy demands are high

Tell me about symbiosis

Most modern eukaryotes evolved from symbiosis. take up oxygen and harness energy from the oxidation of food molecules—such as sug- ars—to produce most of the ATP that powers the cell's activities. It is now generally accepted that mitochondria originated from free-living oxygen-metabolizing (aerobic) bacteria that were engulfed by an ancestral cell that could otherwise make no such use of oxygen (that is, was anaerobic). Escaping digestion, these bacteria evolved in symbiosis with the engulfing cell and its progeny, receiving shelter and nourishment in return for the power generation they performed for their hosts. is partnership between a primitive anaerobic predator cell and an aerobic bacterial cell is thought to have been established about 1.5 billion years ago, when the Earth's atmosphere first became rich in oxygen. that the first eukaryotic cells were formed from an archaeal cell engulfing an aerobic bacterial cell, which would explain the resemblance of archaea to modern eukaryotes, and the mitochondria to bacteria.

Why is there so much non-coding, dispensable DNA?

Much of our noncoding DNA is almost certainly dispensable junk, retained like a mass of old papers because, when there is little pressure to keep an archive small, it is easier to retain everything than to sort out the valuable information and discard the rest.

Termination of translation in bacteria?

Multiple players. Termination codon, UAA, enters A site. Release factors 1 and 2 recognise the termination codon. How? When release factors recognise the termination codon, ribosome dissociates and disassembles. DO NOT CONFUSE TRANSLATION TERMINATION WITH TRANSCRIPTION TERMINATION IN BACTERIA.

Tell me about actin and myosin in muscle

Muscle contraction results from the activity of highly organised arrays of actin filaments and myosin motors. In muscle cells the cytoplasm is filled with parallel arrays of actin and myosin filaments, which use ATP to generate the force to allow them to slide over each other. The actin filaments are attached at their plus ends to regularly placed Z-discs and their minus ends overlap with bipolar assemblies of myosin. The plus end directed movement of myosin leads to shortening of the muscle cell - Actin and myosin filaments generate contractile muscle tissue Bipolar myosin motors pull the actin filaments to cause sliding & contraction

Negative staining

Negative staining allows fine detail to be seen. Macromolecules such as DNA and large proteins can be visualised. molecules are supported on a thin film of carbon and mixed with a solution of heavy-metal salt such as uranyl acetate. After the sample has dried, a very thin film of metal salt covers the carbon film everywhere except where it has been excluded by the presence of an adsorbed macromolecule. Because the macromolecule allows electrons to pass through it much more readily than does the surrounding heavy metal stain, a reverse or negative image of the molecule is created. Negative staining is especially useful for viewing large macromolecular aggregates such as viruses or ribosomes, and for seeing the subunit structure of protein filaments.

Precaution with negative staining and viewing infectious organisms?

Negative staining at both light microscope and electron microscope level should never be performed with infectious organisms unless stringent safety precautions are followed. Negative staining is usually a very mild preparation method and thus does not reduce the possibility of operator infection.

Tell me the typical size of an RBC

Normal RBCs have a diameter of 6 - 8 μm.

Tell me about the nosepiece of the Zeiss microscope

Nosepiece. The stand carries a horizontal limb, to which is attached a rotating nosepiece with four objective lenses (see below). The nosepiece can be rotated by moving the black textured grip. Ensure that the empty position in the nosepiece is directly and vertically above the stage. If in doubt ask your demonstrator.

List the two factors that limit the resolution of electron microscopes other than electron wavelength Tell me the practical limit of resolution of electron microscopes

Numerical Aperture Lack of inherent contrast. 1 to 2 nm

Tell me about bacteriophage T4

One of the most important bacteriophages is T4, which infects and replicates in E. coli. Infection with a single particle of T4 leads to the formation of approximately 200 progeny virus particles in 20 to 30 minutes. The initially infected cell then bursts (lyses), releasing progeny virus particles into the medium, where they can infect new cells. In a culture of bacteria growing on agar medium, the replication of T4 leads to the formation of a clear area of lysed cells (a plaque) in the lawn of bacteria

Tell me about prokaryotic and Eukaryotic ribosomes

P and E have different sized ribosomes, their sizes are measured by their speed of sedimentation in a centrifuge. 70s - 50s and 30 s 80s - 60s and 40s

How do you obtain a finer degree of separation using the ultracentrifuge?

Perform a velocity sedimentation - spin the components in a sucrose density gradient, which increases in density as you move down. The speed at which a component sediments is described by its sedimentation coefficient or S value

Peroxisomes

Peroxisomes are specialised compartments for oxidative reactions (e.g. breakdown of fatty acids)

What is phosphorescence?

Phosphorescence is a type of photoluminescence related to fluorescence. Unlike fluorescence, phosphorescent material does not immediately re-emit the radiation it absorbs. The slower time scales of the re-emission are associated with "forbidden" energy state transitions in quantum mechanics. As these transitions occur very slowly in certain materials, absorbed radiation is re-emitted at a lower intensity for up to several hours after the original excitation. Everyday examples of phosphorescent materials are the glow-in-the-dark toys, stickers, paint, and clock dials that glow for some time after being charged with a bright light such as in any normal reading or room light. Typically, the glow slowly fades out, sometimes within a few minutes or up to a few hours in a dark room

Tell me about channel forming junctions in plants

Plant cells have only one class of intercellular junction, the channel forming plasmodesmata (singular plasmodesma), which like gap junctions directly connect the cytoplasms of adjacent cells. Plasmodesmata bridge the cell wall (~ 0.1µm thick) with cytoplasmic channels 20-40nm in diameter (Figure 41). Running through the channel is a desmotubule, which is continuous with elements of the smooth ER of each cell. The channel itself is formed by continuities between the plasma membranes of adjacent cells. These form as the cell divides; cytokinesis is incomplete at the plasmodesmata. Despite their differences in structure plasmodesmata and gap junctions are functionally similar; they play a role in coordinating the activity of groups of cells (in plants for example during development), the cut off for transfer through plasmodesmata is 800 daltons and permeability is regulated.

Tell me about the culture of Plant cells

Plants that proliferate in culture produce a callus - a mass of undifferentiated cells. Preparation of plant tissue for tissue culture is performed under aseptic conditions under HEPA filtered air provided by a laminar flow cabinet. Living plant materials from the environment are contaminated on their surfaces (and sometimes interiors) with microorganisms, so their surfaces are sterilized in chemical solutions (usually alcohol and sodium or calcium hypochlorite)[1] before suitable samples (known as explants) are taken. The sterile explants are then placed on the surface of a sterile solid or liquid culture medium. Solid and liquid media are composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media are prepared from liquid media with the addition of a gelling agent, usually purified agar. Plant hormones and nitrogen source in the medium (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, excess auxin will result in a proliferation of roots, while excess cytokinin yields shoots. A balance of both auxin and cytokinin will often produce a callus but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As cultures grow, pieces are typically sliced off and subcultured onto new media

Tell me about porins

Porins are a type of membrane protein that forms a beta-barrel pore across the lipid bilayer. Unlike other protein transport channels, the pores from porins are large enough to allow passive diffusion. As a result of the large diameter, porins generally mediate the diffusion of small metabolites, such as amino acids, sugars, and ions. The general structural properties of various porins are the same, regardless of their type. Porins are proteins composed primarily of beta-sheets, connected in the anti-parallel direction. Sixteen to eighteen strands of beta-sheets fold into a cylindrical tube, which is labeled as the beta-barrel. The amino acid sequence is roughly an alternating pattern of non-polar and polar residues, which are positioned in an appropriate way as to generate the tertiary barrel structure. This beta-barrel is located in the lipid bilayer, forming the large hole into the cell. As a result of these interactions, the amino acids facing the outside of the beta-barrel are generally nonpolar, to interact with nonpolar region of the lipid bilayer. However, the inside of the beta-barrel typically contains polar amino acids, to interact with the aqueous environment connecting the two sides of the membrane. Porins are also found in the outer membrane of gram negative bacteria. Gram negative bacteria contain an outer membrane that helps keep out unwanted chemicals, as well as increase virulence. The porins facilitate the diffusion of small molecules and nutrients through the outer membrane and into the periplasm.

Tell me about flagella in prokaryotes

Procaryotes can also be propelled by flagella, which are12-18nm in diameter and made up of a single protein, flagellin, in a helical array. Each flagellum is attached by a short flexible hook at its base to protein discs embedded in the bacterial wall and membrane (Figure 32). These rings form a miniature turbine, which rotates more than 100x/sec, driven by the flow of protons down a gradient across the inner cell membrane. The direction of rotation can change: Counterclockwise rotation leads to forward movement, clockwise rotation causes "tumbling", that randomises direction.

Tell me about pili

Procaryotic cells donʼt have stable junctions but they communicate through pili (latin = hair), short tube-like projections from the cell surface of about 7nm diameter (Figure 37). Pili are anchored into the cell membrane by multiprotein complexes and are used to attach to surfaces. Some pili can generate force and may help bacteria to move. Others (the sex pili) attach to receptor sites on another bacterium and facilitate the transfer of DNA between bacteria.

How many DNA molecules prokaryotes encode their genetic info?

Prokaryotes encode their genetic material in a single DNA molecule However, extrachromosomal DNA(plasmids) can carry additional genetic information

3 Pros of Brightfield Microscopy

Pros: The optics do not change the color of the observed structures. Sometimes stains are used to make certain structures visible. The optics of a bright field microscope do not change these colors. Bright-field optics is generally cheaper than phase contrast optics Bright-field microscopy requires fewer adjustments before one is able to observe the specimens.

3 Pros of Phase-contrast microscopy?

Pros: One of the major advantages of phase contrast microscopy is that living cells can be examined in their natural state without previously being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail. It is possible to visualize certain structures that are otherwise invisible. This includes certain cell organelles which can not be seen well in bright field. Sometimes the phase contrast image subjectively looks better than a bright field image due to the details visible.

translocator protein complexes

Proteins synthesised on the ribosomes of RER feed through translocator protein complexes into the lumen

What's the overall reaction in the formation of acetyl CoA via Pyruvate Dehydrogenase

Pyruvate + CoA + NAD+ --> acetyl CoA + CO₂ + NADH + H⁺

What does the nuclear pore complex do? How many macromolecules per second can NPC transport? In which directions can the NPC transport macromolecules?

Regulates the passage of material in and out of the nucleus through the nuclear pore regulate the passage of molecules larger than about 60,000 Daltons. NPCs can transport up to 500 macromolecules per second and can transport in both directions at the same time. Proteins with nuclear localisation signals are imported into the nucleus, and as we will see later mRNAs and ribosome subunits are exported to the cytoplasm

What is restriction mapping?

Restriction mapping relies on bacterially-derived restriction enzymes which can cut DNA at specific sequences. Each type of restriction enzyme cuts the DNA at a different but specific sequence. When a DNA helix has been cut using these enzymes, the lengths of the fragments can be determined by using gel electrophoresis, which separates the molecules on the basis of size. By using different combinations of restriction enzymes to generate different length fragments, a map can be drawn of the original DNA with the location of each type of restriction site.

How do ribosomes function during protein synthesis

Ribosomal subunits combine, sandwiching an mRNA molecule. smaller subunit provides a framework for protein assembly larger subunit catalyses polypeptide extension. the ribosomal RNA is a ribozyme, catalysing peptide bond formation.

Tell me about light phase

Right the real part of a plane wave moving from top to bottom. Right: the same wave after a central section underwent a phase shift, for example, by passing through a glass of different thickness than the other parts.

Bright Field Microscope:

Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample. Bright-field microscopy is the simplest microscopic technique.

Tell me about thin sections specimen preparation

Samples are fixed to retain their structure, cryofixation and chemical fixation are commonly used. Even a cell normally too thick to be viewed under TEM. The specimen is fixed chemically, dehydrated and embedded in plastic resin. Thin sections are cut using glass or diamond knives in an ultramicrotome. The sections, about 100 nm, are picked up onto grids, and stained using salts of heavy metals which are selectively taken up by different cell components in the section, thereby increasing their electron-scattering power. Grids are viewed in the TEM. Useful for looking at the internal structure of cells and organelles; examination of serial sections allows the construction of models in three dimensions.

Tell me about the methods in subcellular fractionation that can be used to disrupt the plasma membrane that does not destroy the internal components of the cell

Several methods are used, including sonication (exposure to high-frequency sound), grinding in a mechanical homogenizer, or treatment with a high-speed blender. Another method is osmotic shock. All these procedures break the plasma membrane and the endoplasmic reticulum into small fragments while leaving other components of the cell (such as nuclei, lysosomes, peroxisomes, mitochondria, and chloroplasts) intact.

Tell me about the Specimen position of the Zeiss Microscope

Specimen position. The stand carries a mechanical stage, which holds the specimen slide. It is moved backwards and forwards and from side to side by the upper and lower knobs on the mechanical stage adjustment that hangs from the stage on the right hand side.

Tell me about specimen preparation for SEM

Specimens must be dehydrated and covered with a thin, conducting layer (e.g. gold), to prevent electrical charging when subjected to the focuses electron beam and to increase secondary electron production. Hard specimens, such as insect heads, are relatively easy to prepare, but delicate specimens are more difficult.

Tell me about positive and negative staining in electron microscopy

Specimens to be examined by transmission electron microscopy can be prepared by either positive or negative staining. In positive staining, tissue specimens are cut into thin sections and stained with heavy metal salts (such as osmium tetroxide, uranyl acetate, and lead citrate) that react with lipids, proteins, and nucleic acids. These heavy metal ions bind to a variety of cell structures, which consequently appear dark in the final image (Figure 1.32). Alternative positive-staining procedures can also be used to identify specific macromolecules within cells. For example, antibodies labeled with electron-dense heavy metals (such as gold particles) are frequently used to determine the subcellular location of specific proteins in the electron microscope. This method is similar to the use of antibodies labeled with fluorescent dyes in fluorescence microscopy. Negative staining is useful for the visualization of intact biological structures, such as bacteria, isolated subcellular organelles, and macromolecules (Figure 1.33). In this method, the biological specimen is deposited on a supporting film, and a heavy metal stain is allowed to dry around its surface. The unstained specimen is then surrounded by a film of electron-dense stain, producing an image in which the specimen appears light against a stained dark background.

Morphological differences between spermatocytes and spermatids?

Spermatocytes have a large nucleus Spermatids have a phase dark nebenkern and a phase light nucleus

Why is defining a gene difficult?

Supervision

To which organelles do kinesin and dynein bind to?

The ER binds kinesin (+end directed) spreading out into the cytoplasm, The Golgi binds dynein (-end directed) and lies close to the nucleus

where on an amino acid are sugars added

The R group of asparagine and serine

What is a cell?

The basic organisational unit of life

Tell me about cell walls

The cell walls of plants are strong and rigid, providing great mechanical strength but precluding movement. The wall consists of tensile fibers (providing strength) embedded in a matrix (providing resistance to compression). The tensile fibres are polysaccharide chains, polymers of sugars, cross-linked into a network and embedded in pectin, a highly hydrated network of a different polysaccharide. In higher plants the fibres are cellulose, the most abundant organic macromolecule on earth (Figure 5).

Tell me about the kinetochores Tell me about interpolar microtubules

The chromosomes move onto the spindle and are attached to kinetochore microtubules by protein complexes, the kinetochores, at the centromere of each sister chromatid Other interpolar microtubules span between the centrosomes (the spindle poles) to stabilise the spindle. In many cells astral microtubules radiate outwards from the poles and contact the cell cortex to position the spindle in the cell (Figure 34).

Outline the steps of subcellular Fractionation

The first step in subcellular fractionation is the disruption of the plasma membrane under conditions that do not destroy the internal components of the cell. The suspension of broken cells (called a lysate or homogenate) is then fractionated into its components by a series of centrifugations in an ultracentrifuge Fractions obtained from centrifugation are not pure. These fractions are further purified.

Tell me about the Focus of the Zeiss Microscope

The focus controls move the stage. Thus, the distance between the specimen and the objectives (see below) can be controlled and thus you can focus the specimen. There are coarse and fine focussing knobs on each side, so that you can use either hand to control focus. Making sure that the empty position on the nosepiece is above the specimen and that there is plenty of distance between the stage and objectives, observe the microscope from the side and use the two knobs to move the specimen stage.

Transcription factors to regulate gene expression?

Eukaryotic transcription involves more accessory factors than the prokaryotic case. Because of this, transcription can be targeted in many ways by targetting these accessory factors I think? Example showing large usage of accessory factors in eukaryotes: A pre-initiation complex is formed involving various general transcription factors such as TATA-binding protein(binds to TATA box) and TFIIA. These factors are important for recruiting RNA polymerase. Gene specific promoters may also contain regions for the binding of specific inducible(you can increase or decrease the levels of this TF in response to stimuli, so can regulate gene expression this way) transcription factors Eukaryotic RNA polyermase requires general transcription factors(such as TFIIA and TFIIB) to permit basal levels of transcription from core promoter. So these can be targeted to regulate gene expression. Eukaryotic genes also possess transcriptional activators to aid recruitment of the transcriptional machinery and promote transcription. Transcriptional activators can thus also be targeted. Transcriptional activators are bipartite and contain a highly specific DNA binding domain coupled to an activation domain. DNA binding domain may include folds such as zinc fingers. So if you know something that coordinates tightly to zinc, you can regulate transcription this way. Examples of nuclear receptors that respond to hormones: Glucocorticoid receptor and oestrogen receptor, recognise palindromic sequences in DNA. When the hormone binds to this receptor, it results in a conformational change in the transcriptional activator, allowing dissociation from inhibitory chaperones. It can then migrate to the nucleus and bind to DNA recognition sequences(these proteins recognise certain sequences of DNA). The binding permits the activation domain to facilitate transcriptional enhancement.

Tell me about the cytoskeleton in prokaryotes

Even though procaryotic cells are small and lack internal organelles, they also have elements of a cytoskeleton. They have a gene for a tubulin-like protein that can polymerise into filaments and form into a ring (the Z-ring), which is important for cell division (as we shall see, a role carried out by actin in eucaryotic cells). Many bacteria also contain homologues of actin, which assemble into filaments that are important for cell shape and the normal deposition of the cell wall (cf microtubules in plant cells). Actin-like filaments also play a role in pushing apart replicated bacterial plasmids (eucaryotic chromosomes are separated along microtubules). Thus the deployment of actin and tubulin-like filaments in the organisation of the cell appears to be very ancient but the precise roles they play vary between procaryotic and eucaryotic cells. The various structural filaments in the cytoplasm collectively make up the prokaryotic cytoskeleton. Cytoskeletal filaments play essential roles in determining the shape of a bacterium (coccus, bacillus, or spiral) and are also critical in the process of cell division by binary fission and in determining bacterial polarity.

Tell me about the estimated time range for animal cell culture

Even under optimal conditions, the division time of most actively growing animal cells is on the order of 20 hours—ten times longer than the division time of yeasts. Consequently, experiments with cultured animal cells are more difficult and take much longer than those with bacteria or yeasts. For example, the growth of a visible colony of animal cells from a single cell takes a week or more, whereas colonies of E. coli or yeast develop from single cells overnight. Nonetheless, genetic manipulations of animal cells in culture have been indispensable to our understanding of cell structure and function.

Tell me the 3 different vesicle types from the golgi apparatus

Exocytotic vesicles - These vesicles contain protein that are to be sent outside the cell membrane. These vesicles fuse with the plasma membrane, releasing their contents outside the cell. This process is called constitutive secretion. Secretory vesicles - These vesicles are also to be sent outside the cell membrane. However what differs these from exocytotic vesicles is that secretory vesicles need a signal before they are released. When the signal is given, they will fuse with the plasma membrane to release the contents. This process is called regulated secretion. Lysosomal vesicles - These vesicles contain protein that are sent to the lysosome to be digested.

Compound microscope: How do you focus the condenser?

First set the lever which controls the condenser iris to its mid position.The condenser should already be roughly in focus from the adjustment you made earlier (para. 12). Close the field-iris gradually while looking through the eyepiece. At a certain point, you should see a (possibly blurred) image of the hexagonal field-iris come into the field of view. It may well be off-centre. (If you cannot see this and/or you see a grey ring, go on to (c) below.) If you can see it - (a), rack the condenser gently up or down with the condenser focus control to obtain the sharpest possible image of the field-iris. (If it disappears from view as you try to focus, go on to (b).) If you can focus it sharply in this way, do so. Next check that the image of the field-iris is in the centre of the field. If it is not, centre the condenser with the condenser centring controls. Adjust the condenser until the image of the field-iris is exactly centred in the field of view. You may need to close the field-iris slightly as you do this. (b) If, in trying to focus the condenser, the image of the field-iris moves off-centre and disappears from view altogether it means that the condenser is badly off-centre. In this case, open the field-iris until the blurred image of its edge is once again visible in the field of view and centre this as far as possible, using the two condenser centring screws. Focus the condenser to give a sharp image of the field-iris. Centre the condenser exactly if necessary using the condenser centring controls. (c) If you cannot see the edge of the field-iris at all when you close it down, this is because the condenser is very badly out of focus. In this case, leave the field-iris half closed, rack the condenser fully down and then rack it up until the edge of the field-iris is brought into view. Then proceed as in (a) above. The final image of the field-iris must as sharp as possible and exactly centred. This is how we know that the condenser is correctly focussed and centred.

Tell me about Fluorescence Microscopy

Fluorescence microscopy is a widely used and very sensitive method for studying the intracellular distribution of molecules and proteins(Figure 1.28). A fluorescent dye is used to label the molecule of interest within either fixed or living cells. Fluorescence is detected by illuminating the specimen with a wavelength of light that excites the fluorescent dye and then using appropriate filters to detect the specific wavelength of light that the dye emits. Fluorescence microscopy can be used to study a variety of molecules within cells. One frequent application is to label antibodies directed against a specific protein with fluorescent dyes, so that the intracellular distribution of the protein can be determined. Proteins in living cells can be visualized by using the green fluorescent protein (GFP) of jellyfish as a fluorescent label. GFP can be fused to a wide range of proteins using standard methods of recombinant DNA, and the GFP-tagged protein can then be introduced into cells and detected by fluorescence microscopy. can destroy fluorescent proteins by shining very powerful light, fluorescent molecule absorbs light and becomes destroyed enables the observation of specific molecules in real time FRAP experiment,

What are orthologs? What are paralogs What are homologs?

Genes that are related by descent in this way—that is, genes in two separate species that derive from the same ancestral gene in the last common ancestor of those two species—are called orthologs. Related genes that have resulted from a gene duplication event within a single genome—and are likely to have diverged in their function—are called paralogs. Genes that are related by descent in either way are called homologs, a general term used to cover both types of relationship

How to focus the light microscope?

1) Close Field Iris until you see the hexagonal edges. 2) Sharpen the field iris by focusing the condenser focus. Centre it with condenser centering pins. 3) Open field iris until it just disappears from view. 4)Next, adjust the condenser iris. Remove one of the eyepieces and look directly down the barrel of the tube. Open eye piece and open condenser iris using level until 2/3 of eyepiece is occupied by the image. 5)Replace eyepiece.

Outline the 3 steps in which pyruvate is converted into acetyl CoA via the Pyruvate Dehydrogenase Complex

1) Decarboxylation 2) Oxidation 3) Transfer of the resultant acetyl group to CoA

Fluorescent Imaging in Drosophila Embryos. How were these prepared?

1) Embryo fixed with methanol, and washed with PBS-S(PBS + detergent saponin). Detergent probably gets rid of the membrane so antibodies can bind. 2) Embryos incubated with PBS-S containing 5% calf serum(BSA) to "block" unspecific sites to which antibodies might otherwise bind. (I don't know how this works) 3) Embryos incubated with PBS-S-BSA containing a mouse derived primary antibody that binds to Cnn (centrosomal protein) and another primary antibody(rabbit derived) that binds to alpha-tubulin(microtubule protein). Wash again with PBS-S to remove unbound antibody. 4) Incubate embryos with PBS-S-BSA containing anti-mouse secondary antibodies conjugated to a green dye and secondary antibody (Anti-rabbit) conjugated to a red dye. 5) embryos were washed in PBS-S (to remove unbound seconadary antibodies) and mounted in a glycerol based mounting medium containing DAPI(binds DNA and emits blue light under UV light) and an anti-fade agent.

What are the aims of sample preparation?

1) Retain structure 2) Create an image, can be by changing the behaviour of electrons 3) minimise the creation of artefacts

4 methods to regulate eukaryotic gene expression:

1) Splicing 2) small RNAs 3) Chromatin 4) Transcription Factors

What are the approximations of michaelis menten kinetics?

1) The reaction from ES complex to product is irreversible.

Tell me 3 important biological consequences of Actin's interaction with myosin motors

1) stabilises relatively stable structures, for example, in the brush border, microvilli, myosin motors push the membrane to protrude to form microvilli. An important role for actin and myosin in animal cells is to regulate cell shape, both to stabilise permanent structures (such as microvilli in the brush border of our intestinal cells or the hair cells of our inner ear) and to promote cell shape changes. 2) Actin's interaction with myosin promotes movement within cells example: large algal cells Chara corallina, Cytoplasmic streaming is very rapid. Practical class average was 40µm sec-1 3) to promote cell shape changes and movement, in muscle

What are the disadvantages of TEM?

1)TEM's higher resolution comes at a cost: specimen preparation for electron microscopy is complex 2)It is harder to be sure that what we see in the image corresponds precisely to the original living structure(artefacts)

Tell me about the Decarboxylation step in the formation of acetyl-CoA via the Pyruvate Dehydrogenase Complex

1. Decarboxylation: thiamine pyrophosphate (TPP) is combined with the pyruvate and decarboxylated in order to yield hydroxyethyl-TPP. Of the pyruvate dehydrogenase component, TPP is known as the prosthetic group which the carbon atom between the nitrogen and sulfur atoms in the thiazole ring is more acidic than most double bonded carbon groups with pKa values near 10. This reaction is catalyzed by the (E1) pyruvate dehydrogenase component of the multienzyme complex. The carbon center located in the TPP is ionized to form a carbanion which is added to the carbonyl group of pyruvate. As part of decarboxylation, a positive charged ring of TPP stabilizes the negative charge which was transferred to the ring. Finally, the protonation yields hydroxyethyl-TPP. Pyruvate + TPP( coenzyme thiamine pyrophosphate)+ 2 H⁺ --> Hydroxyethyl-TPP + CO₂

Actin microfilament structure?

Globular actin subunits assemble into filaments 2 parallel filaments form a double-stranded helix actin microfilaments assemble in a polarised way, have a + end and a - end. Actin is also polarised: growing more rapidly from the + than the - end Accessory proteins control actin bundling and the formation of complex branched networks

What are the four ways in which new genes can be generated from pre-existing genes

1.Intragenic mutation: an existing gene can be randomly modified by changes in its DNA sequence, through various types of error that occur mainly in the process of DNA replication. 2. Gene duplication: an existing gene can be accidentally duplicated so as to create a pair of initially identical genes within a single cell; these two genes may then diverge in the course of evolution. 3. DNA segments shuffling : two or more existing genes can break and rejoin to make a hybrid gene consisting of DNA segments that originally belonged to separate genes. 4. Horizontal(intercellular)transfer: a piece of DNA can be transfer red from the genome of one cell to that of another—even to that of another species. is process is in contrast with the usual vertical transfer of genetic information from parent to progeny.

Tell me about the oxidation step in the formation of acetyl CoA via the Pyruvate Dehydrogenase Complex

2. Oxidation: In order to form an acetyl group, the hydroxyethyl group which is attached to TTP is oxidized. Simultaneously, the hydroxyethyl group is transferred to lipoamide which is lipoic acid derived that links to the side chain of a lysine residue by an amide linkage. This creates the formation of an energy-rich thioester bond. In this reaction, the disulfide group of lipoamide acts as an oxidant and is reduced to the disulfhydryl form. This reaction is catalyzed by the pyruvate dehydrogenase component (E1) as well and yields the acetyllipoamide. Hydroxyethyl-TPP + Lipoamide --> TPP + Acetyllipoamide

Tell me about the 3 components of the cytoskeleton, and the functions of the cytoskeleton in eukaryotic cells.

3 components: 1) Microfilaments (polymers of actin) 2) Intermediate Filaments 3) Microtubules (polymers of tubulin) cytoskeleton proteins are multifunctional Functions: 1) Cell structure and shape: It is a network of protein fibers supporting cell shape and anchoring organelles within the cell. All three components interact with each other non-covalently. 2) Internal transport of substances Cytoskeleton acts like a rail. Cytoskeleton causes movement of organelles within the cell when necessary plays a role in endocytosis 3) Cytoskeleton is involved in cell division: Microtubules pull paired chromosomes apart. 4) Cytoskeleton is involved in Cell movement: Moving Flagella is due to sliding microtubules surrounded by a membrane. The microtubule arrangement is referred to as a 2X9+2 arrangement.

How does eukaryotic initiation occur without the shine dalgarno sequence?

40s and some eIFS complex, and recognise the 5- capping structure. This helps them find the initiating AUG codon. This releases the initiation factors(Why? Because that is the role of the eIFS, to help start translation by helping the 40s find the AUG codon). Once the eIFS are released, the 60S can now bind, and elongation can now occur. Basically, no shine dalgarno sequence, so instead eukaryotes use the 5' cap to initiate translation. Eukaryotic initiation can also occur in a 5' cap independent manner. This occurs through the use of internal ribosome entry sites. IRES are found in the 5' UTR of mRNA(Why? because I guess this also helps find the AUG start codon, which is always at the 5' end.) How does IRES work? I think the weird RNA structure formed by the IRES can help the ribosome find AUG, similar to capping mechanism??? So IRES is just a cap in a different form, this is brandon speculation.

Which organism servers as a minimal model eukaryote? What are the advantages of using this organism for study rather than other organisms?

A Yeast Serves as a Minimal Model Eukaryote Saccharomyces cerevisiae, the same species that is used by brewers of beer and bakers of bread. To analyze the internal workings of the eukaryotic cell without the additional problems of multicellular development, it makes sense to use a species that is unicellular and as simple as possible. It is robust and easy to grow in a simple nutrient medium. In addition to these features, the yeast has a further property that makes it a convenient organism for genetic studies: its genome, by eukaryotic standards, is exceptionally small.

What is a compound microscope

A compound microscope uses a lens close to the object being viewed to collect light (called the objective lens) which focuses a real image of the object inside the microscope (image 1). That image is then magnified by a second lens or group of lenses (called the eyepiece) that gives the viewer an enlarged inverted virtual image of the object (image 2). The use of a compound objective/eyepiece combination allows for much higher magnification. Common compound microscopes often feature exchangeable objective lenses, allowing the user to quickly adjust the magnification. A compound microscope also enables more advanced illumination setups, such as phase contrast.

Why do eukaryotes require specialized internal organelles whereas prokaryotes require much less of this?

A large surface-to-volume ratio, as seen in smaller prokaryotic cells, means that nutrients can easily and rapidly reach any part of the cells interior. However, in the larger eukaryotic cell, the limited surface area when compared to its volume means nutrients cannot rapidly diffuse to all interior parts of the cell. That is why eukaryotic cells require a variety of specialized internal organelles to carry out metabolism, provide energy, and transport chemicals throughout the cell. Both, however, must carry out the same life processes. Incomplete

Glossary: protist

A protist is any eukaryotic organism that is not an animal, plant or fungus. The protists do not form a natural group, or clade, but are often grouped together for convenience, like algae or invertebrates. In some systems of biological classification, such as the popular five-kingdom scheme(now very much outdated) proposed by Robert Whittaker in 1969, the protists make up a kingdom called Protista, composed of "organisms which are unicellular or unicellular-colonial and which form no tissues." Besides their relatively simple levels of organization, protists do not necessarily have much in common.

Tell me about plant cell totipotency and their subsequent culturing

A striking feature of plant cells that contrasts sharply to the behavior of animal cells is the phenomenon called totipotency. Differentiated animal cells, such as fibroblasts, cannot develop into other cell types, such as nerve cells. Many plant cells, however, are capable of forming any of the different cell types and tissues ultimately needed to regenerate an entire plant. Consequently, by appropriate manipulation of nutrients and growth regulatory molecules, undifferentiated plant cells in culture can be induced to form a variety of plant tissues, including roots, stems, and leaves. In many cases, even an entire plant can be regenerated from a single cultured cell. In addition to its theoretical interest, the ability to produce a new plant from a single cell that has been manipulated in culture makes it easy to introduce genetic alterations into plants, opening important possibilities for agricultural genetic engineering.

What is lens aberration?

Aberration in optics refers to a defect in a lens such that light is not focused to a point, but is spread out over some region of space, and hence an image formed by a lens with aberration is blurred or distorted, with the nature of the distortion depending on the type of aberration.

Tell me about polyribosomes

Arise from the assembly of multiple ribosomes on a single mRNA strand.

Tell me about vibrio cholerae

Has a flagellum can affect the human small intestine and cause cholera related to E-coli

Tell me about hereditary defects in ciliary dynein

Hereditary defects in ciliary dynein cause a syndrome in humans called Kartagenerʼs syndrome. Patients are highly susceptible to lung infections due to paralysed cilia and males are sterile as their sperm are immotile. Interestingly there are also defects in the development of left-right asymmetry of the body axis (i.e. the disposition of internal organs may be reversed); cilia play a role in establishing this axis early in embryonic development.

Tell me about Glycolipids in the membrane Whats the simplest glycolipid Tell me about gangliosides Draw a glycolipid with a sphingosine backbone for me Tell me the functions of glycolipids in the membrane

As the name implies, glycolipids are simply sugar-containing lipids. Glycolipds are typically composed of short, branched chains with less than 15 sugar units. The glycolipids in animal cells come from sphingosine, similar to those in sphingomyelin. As in sphingomyelin, the amino group of the sphingosine backbone is acylated by a fatty acid. Glycolipids have a unit that is linked to the primary hydroxyl group of the sphingosine backbone, which differentiates it from sphingomyelin. In glycolipids, one or more sugars are attached to this group. Glycolipids are arranged in an asymmetric manner with the sugar residues always on the extra cellular side of the membrane. The simplest glycolipid is called cerebroside which contains a single sugar residue. The sugar could be either glucose or galactose. Complex glycolipids, for example gangliosides, contain a branched chain of as many as seven sugar residues. The main function of glycolipids in the body is to serve as recognition sites for cell-cell interactions. The sugar moiety of the glycolipid will bind to a specific complementary carbohydrate or lectin, type of cell-surface protein, of a neighboring cell. The interaction of these cell surface markers initiates cellular responses that contribute to activities such as cell recognition, regulation, growth, and apoptosis. Sphingolipidoses can be associated with defects in metabolism. Another example of glycolipid function within the body is the interaction between leukocytes and endothelial cells during inflammation. Selectins on the surface of leukocytes and endothelial cells will bind to the carbohydrates attached to glycolipids to initiate the immune response. This binding allows for leukocytes to leave circulation and congregate near the site of inflammation. This is the initial binding mechanism, after which it is followed by expression of integrins which form stronger bonds and allow leukocytes to migrate toward the specific site of inflammation. Glycolipipds are also responsible for other immune responses, notably the recognition of viruses within the body. Finally, you know all about the ABO bloodsystem, glycolipids are what differentiates A from B from O

Tell me about the gram positive and gram negative division of bacteria

Bacteria are divided into gram-positive and gram-negative, depending on their staining properties on exposure to a violet dye used in the Gram staining procedure that reacts with the cell wall component, peptidoglycan. Gram-positive bacteria have a single membrane with a thicker cell wall on the outside, readily accessible to the gram stain, while gram-negative bacteria have two membranes surrounding the cell with a thin cell wall lying between them (Figure 4).

Why do tissues have to be cut into very thin sections in electron microscopy?

Because electrons have very limited penetrating power, tissues have to be cut into extremely thin sections (25-100 nm thick)

Tell me about the 5 Complexes involved in the oxidative phosphorylation cycle

Between mitochondrial matrix and intermembrane space, within the innermembrane, there are 5 complexes involved in the oxidative phosphorylation cycle, also known as electron transport chain cycle At complex I - NADH dehydrogenase: NADH is oxidized to NAD+: a process that releases electrons, which is transported by FMN (flavin mononucleotide to the Fe-S center (similar to heme group of hemoglobin) and reduce Q (ubiquinone) to QH2 (ubiquinol). Besides, 4 protons are pumped from matrix to intermembrane space. At complex II - Succinate dehydrogenase: succinate is oxidized to fumarate: a process that releases electrons to reduce FAD to FADH2, which carries the original electrons to reduce Q (ubiquinone) to QH2 (ubiquinol). Again protons are pumped into the intermembrane space At complex III - Ubiquinone Cytochrome c oxidoreductase: Q-cycle transfers electron from QH2 to cytochrome c (cyt c). Again protons are pumped into the intermembrane space At complex IV - cytochrome oxidase: cyt c transfer electrons to oxygen O2 (by the support of Cu-S center and hemes). This is where water is released and proves the role of oxygen in aerobic respiration. Again protons are pumped into the intermembrane space At complex V - ATP Synthase: with protons pumped into intermembrane space now return to the matrix, a rotary complex carries out the job of combining ADP and inorganic phosphate in mitochondria into ATP for cellular energy.

Tell me about channel forming junctions

Both plant and animal cells have channel-forming junctions, which allow the direct passage of molecules between adjacent cells (Figure 40). In animals gap junctions allow the passage of inorganic ions and small water-soluble molecules (maximum pore size ~1-5nm allowing passage up to approx 1000 daltons). The channels are formed by hexameric complexes of transmembrane connexin proteins, which line up between adjacent cells to form continuous channels. This means that cell activities regulated by small molecules can be coordinated (for example to generate the waves of ciliary beating in the lung epithelium). The channels are not fixed in an open state; their permeability can be regulated, often by cytoplasmic levels of calcium ions or pH.

How do you set up the compound microscope under bright field conditions

Brandon: I think you can set up a compound microscope under bright field conditions and phase contrast conditions, I'm not sure double check. For optimal viewing the three lenses and the two irises must be on the same optic axis. The position of the eyepiece is fixed. There is a choice of different objective lenses, and each objective lens can be brought into the beam at a fixed position (however the position is slightly different for each objective; hence the need to make adjustments when you change objective). The relative positions of the eyepiece and objective thus define the optic axis. The condenser lens and the two irises must be aligned with this optic axis. In addition, the sizes of the two irises must be adjusted for optimal conditions of observation and for maximum resolution, with each objective. You will do this.

Tell me about Bulk Transport mechanisms

Bulk transport mechanisms enable large molecules and even larger objects to cross the plasma membrane. Examples of Bulk Transport: The process of exocytosis expels large molecules from the cell and is used for cell secretion. Receptor-mediated endocytosis allows specific large molecules to be taken in by the cell. Other forms of endocytosis allow liquids to be taken in by a process called pinocytosis and large objects, such as debris or microorganisms, to be taken in by phagocytosis.

What denser substitute could you use to replace sucrose in velocity sedimentation?

Caesium Chloride

Tell me about carrier proteins Tell me 3 ways by which carrier proteins can be regulated

Carrier proteins are membrane-bound transport proteins that bind with specific substrates and changes conformation to carry the substrate across the membrane. Facilitated diffusion occurs by use of carrier proteins. There are several types of carrier proteins: uniport carriers, symport carriers, and antiport carriers. Uniport carriers are carrier proteins that move only one kind of substance across the cell membrane. They work by binding to one molecule at a time and transporting that molecule down its electrochemical gradient. Uniport carriers can be regulated by various mechanisms: voltage, physical, and ligand binding. By the voltage regulation, the uniport carrier opens or closes at a critical difference between transmembrane voltage. For the physical regulation, physical pressure will cause the carrier to open or close. Lastly, for ligand binding regulation, ligands bind to the uniport carrier on either the intracellular or extracellular side to induce opening or closing of the carrier. Other carrier proteins can carry more than one molecule across the cell membrane at a time. Symport carriers are carrier proteins that transport two or more different molecules simultaneously across the cell membrane in the same direction. In contrast, antiport carriers are carrier proteins that transport two or more different molecules simultaneously across the cell membrane, of which at least one of the molecules are transported in the opposite direction compared to the others. Generally, these carriers transport at least one molecule down its electrochemical gradient and the other molecules would go against its gradient. The molecules going down the electrochemical gradient will provide the driving potential to push the other molecules against its electrochemical gradient. These types of proteins are classified as secondary transporters, or cotransporters. TransportTypes.png Facilitated diffusion by carrier protein transport is ultimately based on the embedded membrane proteins, in contrast with passive diffusion which occurs everywhere in the membrane. In particular, uniport transport and passive diffusion, both of which describe ion movement down concentration gradients across membranes, differ by their maximum rates: uniport transport is limited to and located at the carrier proteins in the membrane.

Difficulties with observing cells with a light microscope?

Cells are mostly transparent, to be able to see cells, they must be stained. As you will see in the practical it is also possible to exploit the fact that the phase of light passing through a cell is altered by its refractive index to create contrast when the two sets of waves are recombined (phase contrast and differential-interference contrast microscopy - often called Nomarski optics after its inventor). These types of light microscopy can be used to image living cells, often by time-lapse video microscopy as cell movements appear relatively slow. You will get more details and practical experience of various types of microscopy in the practical classes Staining methods can provide the necessary contrast to make intracellular structures visible. Cells usually have to be killed and preserved (by chemical fixation) and are often cut into thin sections before they are stained.

How do cells maintain their integrity?

Cells have a complex internal structure, consisting of organelles, organic macromolecules (such as proteins, nucleic acids, carbohydrates and lipids) and small molecules and ions in solution, contained by the cell membrane. Cells are able to concentrate nutrients from the environment and retain products that they synthesise, while expelling waste products. The cell membrane (also known as the plasma membrane or plasmalemma) maintains the chemical integrity of the cell by keeping separate the cell interior (the cytoplasm) and the external environment.

Tell me about chemiosmosis

Chemiosmosis is the movement of ions across a semi-permeable membrane down its electrochemical gradient. An example of this would be the generation of adenosine triphosphate (ATP) by the movement of hydrogen ions across a membrane during cellular respiration or photosynthesis.

Give an example of a naturally fluorescent molecule

Chlorophyll pigment in chloroplasts is naturally fluorescent and can be observed without any special sample preparation by direct fluorescence detection.

How did chloroplasts originate?

Chloroplasts almost certainly originated as symbiotic photosynthetic bacteria, acquired by eukaryotic cells that already possessed mitochondria

Tell me about cholesterol

Cholesterol Cholesterol is a steroid and they are built from 4 fused hydrocarbon rings. The hydrocarbon tail is connected to the steroid at one end, and a hydroxyl group is connected to the other end. Cholesterol is a steroid important in cell membranes and acts as a precursor to some sex hormones. However, prokaryotes do not have cholesterol. The molecule functions as a buffer or a temperature stabilizer for the membrane in which it can make up of 25% of the membrane. When exist in membranes, the 4 cyclic molecules in the cholesterol molecule lay parallel to the fatty acid chains of the phospholipids, meanwhile the hydroxyl terminal points in the direction with the polar phospholipid heads in which it interact with. The fused ring system of cholesterol means that it is more rigid than other membrane lipids.As well as being an important component of membranes, cholesterol is the metabolic precursor of the steroid hormones. Plants contain little cholesterol but have instead a number of other sterols, mainly stigmasterol and beta-sitosterol which differ from cholesterol only in their aliphatic side-chains.

Size of cilia and flagella?

Cilia and flagella appear as hair-like appendages, about 0.25 µm in diameter, projecting from the cell surface. Flagella are 50-200 µm long and are found on sperm and many protists, allowing them to swim. Cilia are normally shorter (6-10 µm) and produce a beating motion, each cilium slightly out of phase with its neighbour to create wave-like patterns, which can propel cells through fluids, as in swimming protists, or move fluid over the surface of a group of cells, as in our respiratory tract (10⁹ cilia/cm2).

Tell me about Basal bodies

Cilia and flagella are firmly rooted into the cell cortex by basal bodies at their base. Basal bodies have nine groups of triplet microtubules arranged like a cartwheel (Figure 31). They do not arise de novo but by replication of an existing basal body. Basal bodies have a very similar structure to centrioles in the centrosome (see next section) and in some cells, such as the unicellular alga Chlamydomonas, basal bodies and centrioles are functionally interconvertible.

Properties of DNA?

How adapted to its function as an information carrying molecule: antiparallel complementary base pairing order of nucleotides along the strand determines info DNA helix has major and minor grooves Information can also be read by "feeling" the structure of the bases exposed in the major groove base pairing ensures accurate DNA replication - semi conservative replication

Tell me about Confocal Microscopy

Confocal microscopy combines fluorescence microscopy with electronic image analysis to obtain three-dimensional images. A small point of light, usually supplied by a laser, is focused on the specimen at a particular depth. The emitted fluorescent light is then collected using a detector, such as a video camera. Before the emitted light reaches the detector, however, it must pass through a pinhole aperture (called a confocal aperture) placed at precisely the point where light emitted from the chosen depth of the specimen comes to a focus (Figure 1.29). Consequently, only light emitted from the plane of focus is able to reach the detector. Scanning across the specimen generates a two-dimensional image of the plane of focus, a much sharper image than that obtained with standard fluorescence microscopy (Figure 1.30). Moreover, a series of images obtained at different depths can be used to reconstruct a three-dimensional image of the sample. fluorescent light is collected from a single focal plane an optical slice of the cell is visualized without interference from out-of-focus layers. These images can be stored digitally and used to reconstruct a three-dimensional picture of the cell. reduces scattered light being viewed, from out of plane Confocal microscopy can also be used to follow living cells, especially when combined with the use of green fluorescent proteins, and other colored variants, which can be used to "tag" proteins expressed in living cells. This can enable the analysis of protein location, interactions and dynamics in real time, using techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET).

How do you produce very thin sections for electron microscopy?

Dehydrate the specimen Permeate it with a monomeric resin that polymerizes to form a solid block of plastic Cut the block with a fine glass or diamond knife on a special microtome. The resulting thin sections, free of water and other volatile solvents are supported on a small metal grid for viewing in the microscope.

Microscope light and embryo

Don't leave light on when not observing the embryo, to avoid bleaching them.

Tell me about drosophila testes What type of cell division occurs during spermatogenesis.

Drosophila testes are spiral shaped about 2mm long. Contain many cysts of sperm cells at different developmental stages. Both meiosis and mitosis occurs during spermatogenesis.

Tell me about microtubule polarity

Due to the orientation of the dimer subunits, microtubules have a polarity, with a- tubulin exposed at one end (minus end) and b-tubulin exposed at the other (plus end). New subunits are more readily added to the plus end, while subunits tend to be lost from the minus end.

What does the rough endoplasmic reticulum contain? What happens inside the lumen of the rough endoplasmic reticulum? What does the RER do?

ER contains chaperones. These special proteins attach themselves to newly synthesized proteins and assist them in folding into their native conformations. A polypeptide synthesized from ER ribosomes, enters the ER after translation. Although the secondary structure (i.e. beta sheets and alpha helixes) of the newly-synthesized peptide forms almost instantaneously as it is translated,its tertiary structure does not begin to form until it is in the lumen, where it will begin to fold into its specific 3-D conformation with the aid of chaperone proteins [1]. It is inside the lumen of the rough endoplasmic reticulum that N- and O- linked glycosidic bonds are formed between amino acids and sugars, thus this process is called "glycosylation" and the newly formed protein is called "glycoprotein". The endoplasmic reticulum transports the proteins through the use of vesicles.

How big is a mitochondrion?

Each mitochondrion is about 1-10 um long.

Tell me about the purification process of fractions obtained from centrifugation in sub cellular fractionation

The fractions obtained from differential centrifugation correspond to enriched, but still not pure, organelle preparations. A greater degree of purification can be achieved by density-gradient centrifugation, in which organelles are separated by sedimentation through a gradient of a dense substance, such as sucrose. In velocity centrifugation, the starting material is layered on top of the sucrose gradient (Figure 1.38). Particles of different sizes sediment through the gradient at different rates, moving as discrete bands. Following centrifugation, the collection of individual fractions of the gradient provides sufficient resolution to separate organelles of similar size, such as mitochondria, lysosomes, and peroxisomes. Equilibrium centrifugation in density gradients can be used to separate subcellular components on the basis of their buoyant density, independent of their size and shape. In this procedure, the sample is centrifuged in a gradient containing a high concentration of sucrose or cesium chloride. Rather than being separated on the basis of their sedimentation velocity, the sample particles are centrifuged until they reach an equilibrium position at which their buoyant density is equal to that of the surrounding sucrose or cesium chloride solution. Such equilibrium centrifugations are useful in separating different types of membranes from one another and are sufficiently sensitive to separate macromolecules that are labeled with different isotopes. A classic example, discussed in Chapter 3, is the analysis of DNA replication by separating DNA molecules containing heavy and light isotopes of nitrogen (15N and 14N) by equilibrium centrifugation in cesium chloride gradients. picture shows velocity centrifugation

laser scanning confocal microscopy

The image obtained by focusing at any one level in the standard fluorescence microscope is degraded by blurred, out of focus information from the parts of the specimen that lie above and below the plane of focus. Laser scanning confocal microscopy removes out of focus light. Laser scanning confocal microscopy builds an optical section of the specimen. A 3D reconstruction is possible by combining images from different optical sections Like normal fluorescence microscopy this technique can be used to observe events in great detail in live specimens.

Tell me about the Peroxisome

The main function of peroxisomes is to break down long fatty acid chains through beta-oxidation and synthesize necessary phospholipids (such as plasmologen) that are critical for proper brain and lung function. Furthermore, they aid certain enzymes with energy metabolism in many eukaryotic cells as well with cholesterol synthesis in animals. Peroxisomes are also involved in germinating seeds in the glyoxylate cycle, photosynthesis in leaves, and oxidation of amines in various yeasts. Peroxisomes are derived from the endoplasmic reticulum and replicate by fission. This organelle is surrounded by a lipid bilayer membrane which encloses the crystalloid core. The bilayer is a plasma membrane which regulates what enters and exits the peroxisome. There are at least 32 known peroxisomal proteins, called peroxins, which carry out peroxisomal function inside the organelle. The synthesis of plasmalogens in animal cells also takes place in peroxisomes. These organelles are very important to the cell because production of plasmalogens is critical to proper functioning of the nervous system since a lack of plasmalogens causes abnormalities in the myelination of nerve cells. During catabolism of fatty acid chains in animal cells, peroxisomes break down long fatty acids into medium fatty acids which are then transported to mitochondria where the majority of catabolism happens.

Tell me about phospholipids

The major class of membrane lipids are the phospholipids. They are abundant in all biological membranes. Phospholipds are made from four components: one or more fatty acids, a platform to which the fatty acids are attached, a phosphate, and an alcohol attached to the phosphate. The fatty acid portion provides the hydrophobic barrier found in lipids, where as the rest of the molecule has a hydrophilic property, enabling interaction with the aqueous environment. Phosopholipds are built upon a foundation of glycerol, a three-carbon alcohol, or sphingosine. Phospholipids which are derived from glycerol are also known as phosphoglycerides, which consist of a glycerol backbone where two fatty acid chains and a phosphorylated alcohol are attached. The major phosphoglycerides come from phosphatidate through the formation of an ester bond between the phosphate group of phosphatidate and the hydroxyl group of one of several alcohols. Sphingomyelin is a phospholipid found in membranes that is not derived from glycerol. The backbone in sphingomyelin, however, is sphingosine, which is an amino alcohol which contains a long, unsaturated hydrocarbon chain. Phospholipids have a very important property of separating compartments. For example, in evolution, it was very important in dealing with the RNA World Hypothesis. If RNA did not have a phospholipid bilayer, they would not have been able to contain all their chemical and mechanistic reactions in a certain space, and it would have been very hard for these RNA to survive without being disrupted by other arbitrary, outside reactions. Thus, phospholipid bilayers played a significant role in the production and survival of these RNA molecules. Phospholipids have many unique functions. For example, they can function as a reservoir of intracellular protein messengers such as phosphoinositol biphosphate. Phosphoinositol biphosphate is one of the most important secondary messengers in the cell signaling pathway of human. Also, they anchor proteins to cells. This many determines the specific function of that lipid membrane. Phospholipids have a number of other functions making them the most abundant membrane lipid. These include energy storage, cellular shape, and it is a source of acetylcholine. Acetylcholine is a commonly occurring neurotransmitter found in both the peripheral nervous system (PNS) and the central nervous system (CNS). Picture shows phosphodiester bond of phospholipid

Tell me about middle lamella

The middle lamella is a pectin layer which cements the cell walls of two adjoining plant cells together. It is the first formed layer which is deposited at the time of cytokinesis. The cell plate that is formed during cell division itself develops into middle lamella or lamellum. The middle lamella is made up of calcium and magnesium pectates.In a mature plant cell it is outermost layer of cell wall.

Tell me the 3 main components of the compound microscope

The objective lens and eyepiece. The object is viewed by two lenses known as the objective lens and the eyepiece. This arrangement gives two stages of magnification: in the first stage the objective lens forms an image of the specimen at the intermediate image plane, and in the second this image is viewed by the eyepiece (and the eye). The condenser lens. Light from the source is focussed onto the specimen by the condenser lens, to provide high intensity, even illumination so that the highest powers of the microscope can to be used to optimum effect. The irises (also called diaphragms or apertures). There are two irises known as the field iris and the condenser iris, and these limit the diameter of the light passing through the microscope.

Tell me the 6 steps in the Pentose Phosphate Pathway

The pathway has 6 steps: 1) It starts with Glucose 6-Phosphate, Glu 6-P, which comes from other pathways, glycolysis for example. 2) Instead of using the enzyme glucose 6-phosphate isomerase to isomerize the original reactant into fructose 6-phosphate for glycolysis, cells use another enzyme, glucose 6-phosphate dehydrogenase, and one important cofactor, NADP+, to oxidize the Glu 6-P into 6-phosphoglucono-δ-lactone with NADP+ being reduced to NADPH. 3)Next, the enzyme lactonase hydrolyzes 6-phosphoglucono-δ-lactone into 6-phosphogluconate. 4) In this step, cofactor NADP+ is used again as an oxidizing agent to oxidize 6-phosphogluconate to ribulose 5-phosphate in a reaction catalyzed by the enzyme 6-phosphogluconate dehydrogenase with the reduced NADPH as another product and the release of carbon dioxide. 5) Note that in every step of the pathway, an addition of Magnesium cation helps stabilizing the reactions, which involves releases of electrons and protons. 6) In a ketose-aldose reaction catalyzed by the enzyme phosphopentose isomerase, ribulose 5-phosphate is isomerized into ribose 5-phosphate, a precursor for later important reactions, such as DNA synthesis.

What is the purpose of the phosphorylation of glucose to glucose 6 phosphate.

The phosphorylation accomplishes two goals: First, the hexokinase reaction converts non-ionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars. Second, the otherwise biologically inert glucose becomes activated into a labile form capable of being further metabolized.

Tell me about freeze fracture in electron microscopy

The preparation of samples by freeze fracture, in combination with metal shadowing, has been particularly important in studies of membrane structure. Specimens are frozen in liquid nitrogen (at -196°C) and then fractured with a knife blade. This process frequently splits the lipid bilayer, revealing the interior faces of a cell membrane (Figure 1.35). The specimen is then shadowed with platinum, and the biological material is dissolved with acid, producing a metal replica of the surface of the sample. Examination of such replicas in the electron microscope reveals many surface bumps, corresponding to proteins that span the lipid bilayer. A variation of freeze fracture called freeze etching allows visualization of the external surfaces of cell membranes in addition to their interior faces.

Tell me about scanning electron microscopy

The second type of electron microscopy, scanning electron microscopy, is used to provide a three-dimensional image of cells (Figure 1.36). In scanning electron microscopy the electron beam does not pass through the specimen. Instead, the surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan across the specimen. Secondary electrons that are scattered or emitted from the sample surface are collected to generate a three-dimensional image as the electron beam moves across the cell. Image formation does not depend on electrons passing through the specimen, it can be of any thickness. Because the resolution of scanning electron microscopy is only about 10 nm, its use is generally restricted to studying whole cells rather than subcellular organelles or macromolecules.

What is equilibrium sedimentation?

The sedimentation of subcellular components at a density similar to their own. When the density of the subcellular component matches its surroundings.

Tell me about Freeze fracture specimen preparation

The specimen is frozen rapidly in liquid nitrogen. It is then transferred to an evacuated chamber and fractured with a cold knife. A replica of the fractured surface is prepared, it is this which is actually viewed. Useful for looking at the internal structure of cells and membranes (esp, since it freezes the membrane in place, as normally it is fluid), by a method that involves different steps from thin sectioning.

Tell me the stoichiometric cofactors involved in Pyruvate Dehydrogenase Complex

The stoichiometric cofactor includes coenzymes such as CoA and NAD+

Tell me about the centrifugation aspect of subcellular fractionation

The suspension of broken cells (called a lysate or homogenate) is then fractionated into its components by a series of centrifugations in an ultracentrifuge, which rotates samples at very high speeds (up to 100,000 rpm) to produce forces up to 500,000 times greater than gravity. This force causes cell components to move toward the bottom of the centrifuge tube and form a pellet (a process called sedimentation) at a rate that depends on their size and density, with the largest and heaviest structures sedimenting most rapidly (Figure 1.37). Usually the cell homogenate is first centrifuged at a low speed, which sediments only unbroken cells and the largest subcellular structures—the nuclei. Thus, an enriched fraction of nuclei can be recovered from the pellet of such a low-speed centrifugation while the other cell components remain suspended in the supernatant (the remaining solution). The supernatant is then centrifuged at higher speed to sediment mitochondria, chloroplasts, lysosomes, and peroxisomes. Recentrifugation of the supernatant at still higher speed sediments fragments of the plasma membrane and the endoplasmic reticulum. A fourth centrifugation at still higher speed sediments ribosomes, leaving only the soluble portion of the cytoplasm (the cytosol) in the supernatant.

what is glycan

The terms glycan and polysaccharide are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan, even if the carbohydrate is only an oligosaccharide.

Tell me about SEM

The wavelength of electrons is much shorter than that of visible light so electron microscopes have a much higher resolution. Scanning electron microscopy is a direct method for producing a high magnification 3-dimensional image of a specimen, achieved by collecting electrons scattered from its surface. This involves fixing and drying the specimen before coating its surface with a thin layer of a heavy metal. The SEM provides a good depth of field and because the amount of electron scattering depends on the angle of the surface relative to the beam of electrons, the image has highlights and shadows which give it a 3- dimensional appearance.

Tell me about numerical aperture in electron microscopy

Theoretically, this wavelength could yield a resolution of 0.002 nm, but such a resolution cannot be obtained in practice, because resolution is determined not only by wavelength, but also by the numerical aperture of the microscope lens. Numerical aperture is a limiting factor for electron microscopy because inherent properties of electromagnetic lenses limit their aperture angles to about 0.5 degrees, corresponding to numerical apertures of only about 0.01. Thus, under optimal conditions, the resolving power of the electron microscope is approximately 0.2 nm.

Tell me the steps involved in gluconeogenesis

There are 3 basic steps involved in gluconeogenesis Pyruvate to Phosphoenolpyruvate (PEP): The enzyme pyruvate carboxylase converts pyruvate into oxaloacetate by adding the CO2 from the bicarbonate ions with the support of ATP and the coenzyme biotin. Then oxaloacetate is converted into phosphoenolpyruvate (PEP)by the enzyme PEP carboxykinase in a process that uses GTP (guanosine triphosphate) as energy and releases CO2 as a waste product. Note that the released CO2 is actually the same CO2 molecule from the bicarbonate ion at the previous step. The next steps are just the reversed processes of glycolysis going from PEP back to Fructose 1,6-bisphosphate, with released products in glycolysis being reactants/cofactors in gluconeogenesis. Fructose 1,6-bisphosphate (Fru 1,6-P) to Fructose 6-Phosphate (Fru 6-P): This is an irreversible hydrolysis reaction catalyzed by the enzyme Fructose 1,6-bisphosphatase (FBPase-1). This reaction is heavily regulated by Fructose 2,6-bisphosphate. Glucose 6-Phosphate to Glucose: Fru 6-P in the previous step is converted into Glucose 6-Phosphate by reversed step of glycolysis. Glucose 6-phosphate is converted into glucose by a hydrolysis reaction.

Eukaryotic elongation?

There are different components to eukaryotic elonation that must be explored to understand this problem. First we must understand the nature of how the tRNAs are moved into the ribosome. Need to talk about the A site, P site and E site. Elongation is a cyclical process. A site- arrival of aminoacyl-tRNA. P site- formation of the peptide bond. P site has peptidyl transferase activity. (you can bond amino acids to a nucleotide chain, and thus you can transfer nucleotides to a polypeptide chain). Just like initiation of translation has eukaryotic initiation factors, elongation in eukaryotes has eukaryotic elongation factors. The main elongation factors in eukaryotes are eEF1A and eEF2. Out of interest in prokaryotes these are EF-Tu and EF-G. Elongation factors play a key role in eukaryotes. The role of elongation factors are explained in another flashcard.

Tell me about the 4 major classes of potassium channel

There are four major classes of potassium channels: Calcium-activated potassium channel - open in response to the presence of calcium ions or other signalling molecules. Inwardly rectifying potassium channel - passes current (positive charge) more easily in the inward direction (into the cell). Tandem pore domain potassium channel - are constitutively open or possess high basal activation, such as the "resting potassium channels" or "leak channels" that set the negative membrane potential of neurons. Voltage-gated potassium channel - are voltage-gated ion channels that open or close in response to changes in the transmembrane voltage. Potassium channel has two components to it: the filter and the gate. The filter is responsible for choosing the ion, which in this case is potassium, and the gate opens and closes depending on the outside environment such as the effect of voltage or signaled molecules. The structure of the filter is similar to that of hemoglobin in the way that it is a tetramer with four repeating subunits that surround the pore. Also, this structure is known to be asymmetric. Moreover, the subunits are hydrophilic. The flow of potassium ion through the channel happens very quickly. Although the name is potassium channel, it also allows sodium ion to pass through. However, the ratio of potassium to sodium is 10,000 to 1.

Describe the cyclical sequence of events that elongation factors play in bacterial elongation during translation

There are two main elongation factors, elongation factor Tu and EF-G. First it is important to note that elongation factors are not permanent fixtures of the ribosome. They can dissociate and exist independently of it. EF-Tu basically escorts an aminoacyl-tRNA together with GTP towards the A site. This ternary complex leaves if codon not complementary to anticodon of tRNA. IF its complementary, then the GTP gets hydrolysed and EF-Tu quite significantly conformationally changes, and pushes aminoacyl-tRNA onto the P site, and EF-Tu falls away from ribosome. Here in the p site, amino acid gets added to the chain. Then EF-G:GTP comes in, and shifts the ribosome along the mRNA, and releases the tRNA from the P site. The energy to do this comes from GTP hydrolysis.

Tell me about the relative amount of NAD⁺ compared to NADH inside the cell Tell me about the relative amount of NADP⁺ compared to NADPH inside the cell

There is about 1000 times more NAD⁺ compared to NADH There is 0.1 NADP⁺ for every 1 NADPH

Tell me about ligand gated ion channels

These ion channels open or close in response to when it binds to a signal molecule or "ligand." The ligand is not the substance that is transported when the channel opens. Some ions are gated by extracellular ligands and others are by intracellular ligands. Another type of protein channels are ion channels that lets through ions. Many of them are stimulated by electrical or chemical signals and are called gated channels. The stimulants either open the channel or close them. Stimulation of a nerve cell by certain neurotransmitter molecules, for example, opens gated channels that allow sodium ions into the cell. External ligands bind to a site on the extracellular side of the channel. Examples include: - Acetylcholine (Ach) which is the binding of the neurotransmitter acetylcholine at certain synapses. It opens channels that admit sodium ions and initiate a nerve impulse or muscle contraction. - Gamma amino butyric acid (GABA)- binding of GABA at certain synapses allows for the central nervous system to admit chlorine ions into the cell, which inhibits the creation of a nerve impulse. - Calcium channels that permit calcium ions to flow into the membrane. These Ca2+ ions then serve as second messenger internal ligands to initiate various processes, such as activating calmodulin. External ligands may be regulated by enzymes. In neurosynapses, acetylcholinesterase degrades ACh into recyclable components; GABA is degraded in a similar fashion. Ca2+ is removed by active transport via an Na+/Ca2+ pump. These various ligand removal processes help regulate the overall concentrations and restore membrane asymmetry. Internal ligands bind to a site on the intracellular side of the channel exposed to the cytosol. Examples include: - cyclic AMP (cAMP) and cyclic GMP (cGMP) which are both called "second messengers". They are channels that are involved in the initiation of impulses in neurons responding to odors and light The channel that allows chlorine and bicarbonate ions in and out of the cell is regulated by ATP. This channel is defective in patients with cystic fibrosis

What happens after the centriole moves to opposite sides of the cell?

They move to opposite sides of the cell and a new microtubule structure, the mitotic spindle, is nucleated from them.

Outline the 3 main ways to prepare samples for TEM

Thin sections Freeze Fracture Negative Staining

Draw a light microscope

This diagram shows the light path in a standard compound microscope. Light is focused on the specimen by lenses in the condensor. A combination of objective lenses and eyepiece lenses are arranged to focus an image of the illuminated specimen in the eye. Light microscopy allowed cell biology to develop, but light microscopes are limited both by the nature of biological materials - cells are often colourless and translucent, and by the nature of light - diffraction properties mean that the resolution of even the best microscope cannot be better than approximately half the wavelength of illumination light (Abbeʼs law). ADD PICTURE

Tell me about negative staining

This is the most useful for looking at small particles (such as viruses or isolated microtubules). The specimen is suspended in a solution of a heavy metal salt and a drop allowed to dry down on an EM grid already covered with a thin supporting film. Contrast arises between the unstained specimen and the electron dense surrounding, dried stain. Basically, you are contrasting a thin specimen with an optically opaque fluid

Tell me about glycosylation

This is the process of adding sugar to a molecule. Glycosylation happens in the golgi apparatus and in the endoplasmic reticulum. Glycosylation can only happen when an amino acid group with a hydroxyl (-OH) attaches to a sugar molecule. Through a condensation reaction (water leaving) a sugar is added to the amino acid.

Tell me about Pyruvate Dehydrogenase Complex

This process involves with the conversion of pyruvate molecule into compound called acetyl coenzyme A, or acetyle CoA. This step is the junction between glycolysis and the Krebs Cycle (Citric Acid cycle) and is accomplished by a multi-enzyme complex that catalyzes three reactions: Pyruvate's carboxyl group (COO-), which is fully oxidized and is removed to release CO2 The remainning two-carbon is oxidized and form a compound named acetate. An enzyme transfers the extracted electrons to NAD+, storing energy in the form of NADH Finally, coenzyme A(CoA), a sulfur-containing compound derived from a B vitamin, is attached to the acetate by an unstable bond and this makes the acetyl group become very reactive. acetyl CoA has a high potential energy will undergoes the Citric Acid cycle to release energy to make ATP. Pyruvate + CoA + NAD⁺ → acetyl CoA + CO₂ + NADH + H⁺ This is an irreversible reaction which links glycolysis and the citric acid cycle together.

Examples of uses of laser scanning confocal microscopy

To see clearly the arrangement in dividing cells of chromosomes (fluorescent dye binds to DNA) and microtubules (fluorescent dye bound to an antibody that binds to the protein tubulin)

Where are ribosomes tethered to?

To the nuclear membrane and RER

Give an example of attenuation in bacterial gene regulation

Trp operon is a good example of attenuation. Key players in the Trp operon: 1) Tryptophan 2) Attenuator sequence 3) Trp operon The concentration of tryptophan in the cell determines the existence of an attenuator on the mRNA. Attenuation controls gene regulation at the translational stage. The attenuator is a secondary structure of RNA. Attenuators are usually located between the promoter and the first gene of the operon. (Makes sense because located after the promoter means its transcribed, but before the first gene means it can act as a regulator BEFORE the first ORF is translated. ) Attenuators often involve a short leader sequence(Why called leader sequence? Maybe its because its the "leader" in the sense that its the first thing thats on the mRNA. And it controls whether the rest of the mRNA is translated or not.) At high levels of Trp, ribosome rapidly translates the leader sequence which causes the formation of a specific termination hairpin, that causes the RNA polymerase to fall off. If levels of Trp are low, RNA polymerase pauses, and because it pauses, a different RNA structure forms, and then RNA polymerase continues transcribing. Trp operon is also under the control of a repressor protein. Repressor and attenuator together enable a 700 fold variation in expression of the Trp operon.

Tell me about two-photon excitation microscopy

Two-photon excitation microscopy is an alternative to confocal microscopy that can be applied to living cells. The specimen is illuminated with a wavelength of light such that excitation of the fluorescent dye requires the simultaneous absorption of two photons (Figure 1.31). The probability of two photons simultaneously exciting the fluorescent dye is only significant at the point in the specimen upon which the input laser beam is focused, so fluorescence is only emitted from the plane of focus of the input light. This highly localized excitation automatically provides three-dimensional resolution, without the need for passing the emitted light through a pinhole aperture, as in confocal microscopy. Moreover, the localization of excitation minimizes damage to the specimen, allowing three-dimensional imaging of living cells.

Tell me about uniporters and tell me a couple of examples

Uniporters are able to transport a specific species like ion channels in either direction governed only by concentrations of that species on either side of the membrane. Can be either a channel or carrier protein, uniporter is an integral membrane protein involved in facilitated diffusion. Uniporter carrier proteins bind to one molecule of solute at a time and transport it with the solute gradient. Uniporter channels open in response to a stimulus and allow the free flow of specific molecules. Since uniporters may not utilize energy other than the solute gradient, they may only transport molecules along with the solute gradient, and not against it. It is good to note that with uniporters the rate of movement is much higher than passive diffusion because the molecule never comes in contact with hydrophobic core of the membrane. Examples: GLUT1 - widely distributed glucose transporter Glucose uniporter shuttles between two conformational states. The net flow is reversed when the concentration of glucose changes. GLUT4- primary insulin regulated glucose transporter in muscle and adipose tissue Potassium leak channels are also regulated by voltage and help restore the resting membrane potential after impulse transmission.

How do you distinguish between different molecules using fluorescence in a cell?

Use different fluorophores, as different fluorophores have different spectral properties and therefore emit light of different colours, so several molecules can be observed and distinguished in the same cell

How do you initiate Translation?

Use shine dalgarno sequence (applicable for prokaryotes only) The 16s rRNA must bind to the shine dalgarno sequence, which is 6 to 8 nt before the initiating AUG codon. (its to help position the ribosome to start translation).

Tell me about SIM and STED

Very newly developed technologies such as structured illumination (SIM) and stimulated emission depletion (STED) even circumvent the theoretical resolution limit of the light microscope - allowing visualization of nanometer sized structures inside living cells.

Why do samples have to be dehydrated in electron microscopy?

When in vacuum, causes rapid evaporation of water which will distort the biological tissue inside.

What happens when the 50S subunit is recruited to the 30S-mRNA complex?

When shine dalgarno and the entire RNA binds to 30s subunit, THEN the 50S subunit is recruited I think. 50S subunit results in GTP hydrolysis by IF2, and release of the IFs. What is the purpose of GTP hydrolysis? Provides energy for something. IF2 brings the initiator tRNA towards the 30S ribosome complexed with the mRNA. hydrolysis of GTP results in binding the massive 50S structure towards the 30S-mRNA complex I think. This process occurs in Prokaryotes

Tell me the 4 components of ATP synthase

While the electron transport chain transports electrons and pumps H+ ions into the intermembrane space, the process of ATP synthesis does not occur until the ATP Synthase. The other part of the oxidative phosphorylation, after the electron transport chain, is the ATP synthase. ATP synthase is a transport protein that is consists of four parts, the Stator, the Rotor, the internal rod, and the Catalytic knobs. The H+ ions in the intermembrane space pumped by the electron transport chain will flow down their gradient first through the stator. The stator is anchored in the membrane. The H+ ion then binds onto the rotor, which is shaped somewhat like a waterwheel. This binding causes the rotor to change its shape and thus, makes it spin within the membrane. The spinning of the rotor causes the internal rod to spin, which leads the catalytic knob to spin. The spinning of the catalytic rod causes the catalytic sites in the rod to transform ADP and inorganic phosphate group into ATP in the mitochondrial matrix. Overall, the ATP synthase functions like a waterwheel. When the concentration of H+ ions in the intermembrane space becomes higher than that of the matrix, the H+ ions will go down the ATP synthase and facilitate the rotor of the ATP synthase. This causes the other components of the ATP synthase to spin. As a result, ATP can be generated from the catalytic sites in the catalytic rod.

Tell me about phase-contrast microscopy

a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells (usually in culture) the phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. PCM converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.

Define cilium

a hair-like locomotor organelle of eucaryotic cells, containing a core of microtubules. Similar to a flagellum but shorter, more numerous and with a different type of beat

Define chromosome

a single large DNA molecule associated with protein in the nucleus of eucaryotic cells. Sometimes used to refer to the DNA molecules of bacteria, mitochondria and chloroplasts

Glossary: thylakoid

closed membrane system of chloroplasts containing the photosynthetic energy transduction system. Also found in cyanobacteria, photosynthetic procaryotes

Tell me about breaking the resolution barrier in fluorescence microscopy

confocal and fluorescence microscopy are still limited by the resolution limit of the light microscope ie about 200nm. However, techniques are being developed constantly to overcome this barrier, they use variations on fluorescence microscopy. 3 methods are: STED (Stimulated Emission Depletion Microscopy) PALM (Photo-Activated Localization Microscopy) SIM(Structured Illumination Microscopy) These techniques can reach resolutions of about 50nm.

Tell me about the organisation of cilia and flagella when you take a cross section

cross section of cilia and flagella - have common organisation Cilia and flagella have a common organization in which pairs of microtubules (called doublets) extend their entire length in a 9+2 configuration (Figure 30). At regular intervals along their length accessory proteins link the doublets together. Ciliary dynein forms bridges between neighbouring doublets and motor activity between them produces a sliding force between adjacent doublets. The presence of other links prevents sliding and the dynein force is converted into bending.

tell me about where you can find polyribosomes

endoplasmic reticulum can have polyribosomes attached polyribosomes also exist free in the cytoplasm

Why do eukaryotes have more internal membranes

eukaryotes have a large volume to SA ratio, so need more internal membranes to increase SA:vol raito

Tell me the differences in locomotor organelles between eukaryotes and prokaryotes

eukaryotic cell - Eukaryotic cells may have flagella or cilia. Flagella and cilia are organelles involved in locomotion and in eukaryotic cells consist of a distinct arrangement of sliding microtubules surrounded by a membrane. The microtubule arrangement is referred to as a 2X9+2 arrangement prokaryotic cell - Many prokaryotes have flagella, each composed of a single, rotating fibril and usually not surrounded by a membrane (see Fig. 10). There are no cilia.

Tell me about the differences in Cell Wall between prokaryotes and eukaryotes

eukaryotic cell a. Plant cells, algae, and fungi have cell walls, usually composed of cellulose or chitin. Eukaryotic cell walls are never composed of peptidoglycan b. Animal cells and protozoans lack cell walls prokaryotic cell a. With few exceptions, members of the domain Bacteria have cell walls composed of peptidoglycan b. Members of the domain Archae have cell walls composed of protein, a complex carbohydrate, or unique molecules resembling but not the same as peptidoglycan.

Tell me the general differences in membrane between eukaryotes and prokaryotes

eukaryotic cell a. The cytoplasmic membrane (see Fig. 2 and Fig. 3) is a fluid phospholipid bilayer (see Fig. 5) containing sterols (see Fig. 6) . b. The membrane is capable of endocytosis (phagocytosis and pinocytosis) and exocytosis . prokaryotic cell a. The cytoplasmic membrane (see Fig. 4); is a fluid phospholipid bilayer (see Fig. 5) usually lacking sterols. Bacteria generally contain sterol-like molecules called hopanoids b.The membrane is incapable of endocytosis and exocytosis.

Tell me the general differences between the nuclear body of eukaryotic and prokaryotic cells

eukaryotic cell a. The nuclear body is bounded by a nuclear membrane having pores connecting it with the endoplasmic reticulum (see Fig. 2 and Fig. 3). b. It contains one or more paired, linear chromosomes composed of deoxyribonucleic acid (DNA) associated with histone proteins ). c. A nucleolus is present. Ribosomal RNA (rRNA) is transcribed and assembled in the nucleolus. d. The nuclear body is called a nucleus a. The nuclear body is not bounded by a nuclear membrane (see Fig. 4). b. It usually contains one circular chromosome composed of deoxyribonucleic acid (DNA) associated with histone-like proteins. c. There is no nucleolus. d. The nuclear body is called a nucleoid .

Tell me generally the differences of cell division between eukaryotic cells and prokaryotic cells

eukaryotic cell a. The nucleus divides by mitosis . b. Haploid (1N) sex cells in diploid or 2N organisms are produced through meiosis . prokaryotic cell a. The cell usually divides by binary fission . There is no mitosis. b. Prokaryotic cells are haploid . Meiosis is not needed.

What is the initiator tRNA?

formylated Met-tRNA. Formylation is adding an aldehyde group

Central Dogma of Biology?

francis crick, who mentioned central dogma? use in essays RNA CODES for amino acid, does not make

Myosin family

interact with actins. Myosin family are closely related to kinesins. the majority are plus end directed motors

Tell me the structure of the nuclear pore complex How many nuclear pores does the typical mammalian nucleus possess?

made up of about 30 different NPC proteins, 3000 - 4000 pores

How do you find out if a plasma membrane protein spans the membrane?

membrane protein extracted and treated with proteases, peptides separated and radioactive peptides * detected by autoradiography

Glossary: spindle apparatus

microtubular structure for the alignment and movement of chromosomes during cell division in eucaryotic cells

Where do microtubules grow out from?

microtubules radiate from a central point - the centrosome. The centrosome is made from a ring of microtubules. the minus end of the microtubules, originate at the centrosome. Polarised network permeates the cell In animal cells microtubules grow out from a microtubule organising centre (MTOC) - the centrosome MTOC lies centrally in the cell, close to the nucleus The minus ends are embedded and stabilised in the centrosome Plus ends radiate out into the cytoplasm Brandon : perhaps + end on tip because membrane phospholipid heads are negative, so there is an attraction between the 2.

mitochondria and chloroplasts - organelles specialised for what common function?

mitochondria and chloroplasts - organelles specialised for energy transduction Phototransduction occurs on grana, stacks of thylakoid membranes

Arabidopsis thaliana

model plant

Tell me a limitation of the culture of normal fibroblasts

most normal cells cannot be grown in culture indefinitely For example, normal human fibroblasts can usually be cultured for 50 to 100 population doublings, after which they stop growing and die.

tell me about kinesin and dynein movement, and their speeds

movement of kinesin and dynein per atp hydrolysis is small, but atp hydrolysis frequency is very large, so can move relatively fast. Each step is a few nm. Movement can reach 2-3µm sec-1 (kinesins) or 14µm sec-1 (dyneins) Hydrolysis of ATP produces a conformational change in the motor protein generating the force necessary for movement and also regulates a cycle of motor protein binding and release from the microtubule.

Formula for resolving power of light microscope?

note the units for the wavelength of light is in µm

Why is it called chemiosmotic COUPLING?

proton gradient COUPLED to ATP synthase

Multiple internal compartments in eukaryotic cells maximise metabolic efficiency by...?

providing additional membrane area to act as a reaction platform creating environments in which high concentrations of specific reagents can be held at a precise pH

Compound microscope: What do you do in order to see an image of the condenser-iris?

remove one of the eyepieces

Production of sperm

several rounds of cell division prior to elongation and development of sperm cells.(mitosis) A stem cell divides producing a new stem cell and a gonial cell(asymmetric division). Gonial cell undergoes 4 rounds of incomplete MITOSIS to produce a cyst of 16 interconnected primary spermatocytes. Spermatocytes mature over several days, increasing their size and duplicating their centrioles in preparation for meiosis. Centrioles also elongate ~ 10-fold, in preparation for their future role as basal bodies, making them resolvable by standard light microscopy(because they became so big). Two meiotic divisions of the spermatocytes produce a cyst of 64 haploid round spermatids(round because no tail yet)(so spermatocytes give rise to spermatids). Round spermatids elongate as they develop into mature sperm. Following these meiotic divisions, the spermatids each contain one of the four centrioles that were originally present in the spermatocyte. This centriole later becomes the basal body from which the sperm axoneme is organised.

epimer

single chiral carbon centre changed

Glossary: glyoxysome

single membrane bound organelle containing enzymes of the glyoxylate cycle

What structures do cells use to accomplish transduction of energy? Essay plan

tbc

Tell me about the Golgi Apparatus's structure Tell me about the Golgi Apparatus's functions

the structure is made of stacks of flattened membranous sacs, or cisternae. Unlike the cisternae of the endoplasmic reticulum or ER, these membranes are not connected. Different layers in golgi cisternae possess different resident enzymes The Golgi apparatus is responsible for the processing and packaging of protein and lipids, as well as processing proteins for secretion. The Golgi apparatus is often thought of as a post office; through the process of protein glycosylation, sugars are added to the protein which dictate to where the protein should travel. N-linked glycosylation is begun in the endoplasmic reticulum but is continued in the golgi complex. O-linked glycosylation is solely done in the golgi complex. After proteins have been modified in the golgi apparatus, they are usually sent to either the lysosomes, secretory granules, or plasma membrane depending on the signals encoded within the protein sequence and structure. For this reason, Golgi complex is recognized as "the major sorting center" of the cell.

Give evidence that eukaryotic cells may have originated as predators

their cells are, typi- cally, 10 times bigger in linear dimension and 1000 times larger in volume. they have an elaborate cytoskeleton - gives cell mechanical strength, controls its shape, controls its movements Has internal membranes involved with secretion and digestion Lack a tough cell wall of most bacteria, animal cells and protozoa, can change shape rapidly and engulf other cells and small objects by phagocytosis Nuclear membrane - It may also require that the cell's long, fragile DNA molecules be sequestered in a separate nuclear compartment, to protect the genome from damage by the movements of the cytoskeleton.

Diptheria toxin and cholera toxin

these are both enzymes that catalyse ADP ribosylation(and inactivation) of key cellular enzymes or proteins.

Tell me about Transmission electron microscope

transmission electron microscopy is similar to the observation of stained cells with the bright-field light microscope. Contrast in the EM results from the scattering of electrons, so cells are stained with heavy metal salts (of osmium, lead and uranium) to create electron-dense stained regions, which scatters electrons, providing contrast. Electrons pass through unstained regions and are focused to form an image on a fluorescent screen. As electrons would be scattered by collisions with molecules in the air, specimens must be viewed in a vacuum. Electrons travel down the column through a series of electron lenses (electromagnets). Electrons that encounter a heavy metal ion as they pass through the sample are deflected and do not contribute to the final image, so stained areas of the specimen appear dark. Contrast in the image arises when different regions of the specimen transmit different numbers of electron. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a charge-coupled device. CCDs allow live imaging. Cells have to be fixed for electron microscopy and as electrons have only very limited penetrating power, extremely thin sections of cells embedded in plastic (50-100nm thick) must be cut for viewing. Clearly the preparation of material for EM is likely to introduce artifacts; working out which of the structures seen in the EM are real and which are artifacts has been a source of controversy since the introduction of this technique. Labelling of specific structures is possible in the EM, using antibodies (which bind to specific proteins in the cell) attached to gold particles. Sometimes, sections dont go through all the organelles, so sometimes in a section of a cell, you wont be able to see the nucleus

Monomers of microtubules

α and β tubulin associate, then form protofilaments (head to tail) 13 protofilaments of tubulin heterodimers (α to β) assemble into hollow cylinders (α to α and β to β) Filaments are polarised: Subunits are added more readily at the plus end and lost from the minus end Microtubules are hollow cylinders about 25nm in diameter made up from a subunit which comprises a heterodimer of two closely related proteins, α- and β-tubulin. These subunits bind to each other head-to-tail (an α to a β) to form protofilaments, which then assemble through side-to-side interactions (α - α, β -β) into hollow cylinders of 13 parallel protofilaments (Figure 26).