Microbial Biotechnology

Last post about applications! It has a smart-sounding title too :)

Be able to define biotechnology, bioremediation (natural and artificial), biosensors, biopesticides/bioinsecticides, bioreactors.
Understand the basic principles and uses of the above.

Biotechnology is basically using microorganisms to do stuff. Yay :)

Bioremediation

One form of biotechnology is bioremediation, which is essentially using microorganisms to fix some kind of environmental problem. Some microbes eat up toxic compounds so they are good for cleaning up things. For example, in the Exxon Valdez oil spill of 1989, nitrogen and phosphorus fertiliser was added in order to increase the number of hydrocarbon-degrading bacteria. This helped significantly in cleaning up the spill. There are two main types of bioremediation: natural, in which organisms already present are encouraged to speed up their degradation activities (as in the case of the oil spill), and artificial, in which microorganisms are genetically modified in order to give them the ability to degrade toxic compounds. Unfortunately there are currently no major success stories in artificial bioremediation- perhaps organisms genetically modified in the laboratory are not as well equipped to survive out "in the wild."

Biosensors

Another form of biotechnology is biosensor technology. Biosensors have two components: a biological component, which senses stuff, and a physicochemical component, which converts whatever the sensor has found into a signal that can be detected. Some biosensors used to detect pollutants are based on luminescent cells. For example, luminescent E. coli can be used to test for pollutants- if alive, they will emit light, but not if they are dead.

Biopesticides/Bioinsecticides

In this form of biotechnology, microorganisms are used to kill pests. Toxin genes from these microorganisms can also be cloned into plants to kill the pests that would otherwise eat them. In turn, this cuts down on the amount of pesticide that needs to be sprayed on crops to keep them healthy.

Bioreactors

Bioreactors, also known as fermenters, are large vessels in which microorganisms can be farmed for industrial use. They have sensors to provide information and pumps in which oxygen and nutrients can be supplied.

Be able to list major industrial products made by recombinant bacteria and their uses.

This is only a short list just to give you an idea- it's definitely not exhaustive.

• Cotton- Often produced by plants with a Bt gene (one of the genes that produces a protein that is toxic to pests).
• Jeans- The faded look on jeans is often produced from cellulase, which loosens dye particles form the cellulose fibres of cotton. Cellulase genes from fungi are cloned into bacteria. The dye itself is produced from indole, which in turn is converted to indigo through the use of a gene cloned from Pseudomonas to E. coli.
• Some plastic- Some bacteria produce polyhydroxyalkanoate granules, which may provide a more environmentally friendly alternative to plastic.
• Hormone replacement products
• Antibiotics and vaccines
• Laundry detergent- Often contains enzymes such as amylase and protease in order to remove specific stains.

Genetic Engineering of Plants

Yet another post on the applications of cell biology! Hopefully this post will also help you understand some of the science behind GMOs.

Be able to define a transgene.

A transgene is simply a gene that has been inserted into the genome of an organism. This can be done through recombinant DNA technology, which I touched on in an earlier post.

Know the properties of Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a bacterium that infects plants, forming small galls, or tumours. It can be used to introduce transgenes into plants as they contain Ti plasmids, as I will explain next.

Be able to describe a Ti plasmid and the properties that make it useful for genetically modifying plants.

Ti plasmids, or tumour-inducing plasmids, are the plasmids inside Agrobacterium tumefaciens. (I've described plasmids before in my post about cloning.) Ti plasmids contain a section called T-DNA, which can be integrated into a plant chromosome. Within this T-DNA is the onc gene, which contains the information required for the formation of a gall. Elsewhere in the plasmid is the vir gene, which is required for the transfer of T-DNA.

Know the basic steps in regenerating a plant from transformed callus tissue.

The production of transgenic plants often involves the binary vector system, in which two plasmids are used. One of these plasmids has T-DNA but no vir gene. This plasmid has the foreign DNA inserted into the T-DNA section- a process also described in that post about cloning. The other plasmid has the vir gene but no T-DNA. Both plasmids are required for insertion of the transgene.

To insert the gene, small discs taken from leaves of a plant are incubated in a dish along with genetically engineered Agrobacterium tumefaciens. These bacteria infect the edges of the discs, as this is where wounded cells are located. Calluses then grow here. To ensure that calluses all contain the new gene, they are usually grown on some kind of selectable media- for example, if an antibiotic-resistance gene is to be transferred over, the calluses will be grown on an antibiotic-containing media. The medium also contains plant growth substances to encourage the growth of shoots. These shoots can be transferred to a root-inducing medium where they can grow to full size.

Another way in which a transgene can be inserted is through biolistic genetic engineering, which is a fancy way of saying "shooting it with a gene gun." In this method, transgene-coated particles are shot into the plant tissue, allowing DNA to become incorporated into the plant chromosome.

Be able to define sense and antisense plants.

Sense plants are genetically modified plants that either produce a new protein or produce more of a protein that they already produce. Antisense plants, on the other hand, produce less of a target protein.

Molecular Cell Biology and Disease

In these next few posts, I'll start getting into the applications of cell biology. This is all just getting our feet wet though- these following topics are the kinds of things that some people spend their whole lives working on.

Outline some of the major causes of molecular diseases.

There are lots of causes of molecular diseases. Cells are quite complex, and if any of the many processes that take place within the cell don't work properly, the consequences can range from benign to disastrous. Factors that can cause these processes to screw up in the first place include genetics, infectious and chemical agents and direct trauma.

Here are a few of the problems and diseases that can result:

• Structural molecular problems. These include prions (infectious misfolded proteins) such as those that cause Mad Cow's Disease and denaturation of other proteins due to chemical exposures.
• Enzyme function problems. These can include overactivity/underactivity of enzymes, the blocking of enzymatic pathways or a complete absence of particular enzymes altogether.
• Cell signalling problems. These include neurochemical uptake issues (as is possibly the case for many kinds of mental illness) and hormonal imbalance (as in the case for gigantism and dwarfism).
• Cell membrane transport problems. These are involved in cystic fibrosis, as I shall discuss later.

Describe the molecular causes of several example diseases.

Malaria

Malaria is caused by a parasite that infects mosquitoes. Mosquitoes can then pass the parasites onto us when they bite us. The parasites enter our liver cells and reproduce, making liver cells burst. They also infect red blood cells, making them eventually pop. Hence some very severe consequences can often result from malaria, such as weakness, anaemia and death. Malaria is a serious disease, which is why the 2015 Nobel Prize in Physiology or Medicine was awarded to Tu Youyou, who discovered Artemisinin, currently the most effective drug for treating malaria.

So what actually happens within red blood cells to make them pop? Well, once inside red blood cells, the parasites start making lots of changes to make themselves at home. They form small organelles called Maurer's clefts, which fuse to the membrane. They then activate secretory pathways and modify the membrane in several different ways. One of these modifications is the insertion of channels, which increases the permeability of the membrane to various substances, which in turn also increases the rate of lysis.

Cystic Fibrosis

Cystic fibrosis is a disease with genetic origins. It involves a mutation in the CFTR gene, which codes for Cl- pumps in the cell membrane. Since less Cl- can exit the membrane, less water follows by osmosis, and so the mucus that lines body cavities becomes thicker than usual. This can have further implications, such as being unable to breathe properly due to thick mucus in the lungs.

Mind you, overactive Cl- pumps are an issue as well. When too much water is lost, death can easily result from dehydration. It has been suggested that people with one allele for cystic fibrosis may be less at risk for death due to diarrhoea and dehydration in diseases such as cholera.

Muscular Dystrophy

Muscular dystrophies are diseases in which the muscle cells are fragile and therefore more likely to rupture and die. I have some pictures of muscle cells from both healthy and mdx mice (mdx mice are basically mice with muscular dystrophy, used for studying the disease) on an earlier post about muscles.

One fatal type of muscular dystrophy is Duchenne muscular dystrophy (DMD). It's an X-linked disease, so guys are more likely to have it than girls. It is thought that 30% of cases result from spontaneous mutations. Muscles get progressively weaker, to the point where death results from heart or respiratory muscles becoming too weak to support the patient. In Duchenne muscular dystrophy, a protein called dystrophin is missing completely. Dystrophin is a cytoskeletal protein that attaches actin to the plasma membrane. Without dystrophin, there is an increased susceptibility to muscle damage and impaired healing after damage.

Not all muscular dystrophies are fatal, however. (Okay, well, everyone's going to die eventually, but you know what I mean.) Becker muscular dystrophy (BMD) is a much milder form of muscular dystrophy that is not usually fatal. In BMD, dystrophin is present, albeit much shorter than normal, allowing for at least partial function.

Protein Function

Once again, this post is included mainly for completeness.

Know proteins bind other molecules and be able to give examples of these other molecules.

I think I've mentioned this before. This binding is why protein shape is so critical to its function- see my earlier post on levels of protein structure. As an example, antibodies bind to specific molecules called antigens, targeting them for destruction.
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Be able to give an example of fibrous and globular proteins.

Many structural proteins, such as keratin, are fibrous. I'm not sure what the exact definition of a fibrous protein is but the idea I'm getting is that they are long and ropelike, and can often wind around each other into coiled-coils for even greater stability.

Many enzymes are globular proteins. Globular proteins are more "folded up" and usually have their hydrophobic residues on the inside and hydrophilic residues on the outside. There is also a special "active site" which is where the ligand (another molecule) binds.

Know how enzymes are regulated and be able to give examples.

Enzymes are regulated through several different mechanisms. Their production can be regulated via regulation of gene expression. Degradation, too, can also be regulated. Enzymes can also be compartmentalised in different areas of the cell- for example, DNA endonuclease normally exists in the cytosol, but when it's time for the cell to die, it moves into the nucleus where it chops up the DNA. Another way in which enzymes can be regulated is through the binding of other molecules. Sometimes binding of other molecules alters the shape of an enzyme, which may change the shape of the active site, which in turn may affect the ability of the enzyme to carry out its function. (Proteins that are regulated like this are known as "allosteric.") Addition or removal of phosphate groups may also contribute to the regulation of enzymes.

Know how to interpret an evolutionary tree.

As there are evolutionary trees of organisms, there are also evolutionary trees of proteins which reflect the similarity of amino acid sequences. Proteins that cluster together are more similar to each other, whereas those that are further apart are less similar. Proteins that are similar in amino acid sequences also tend to be similar in function.

Know how motor proteins generate movement.

Motor proteins generate movement by changing their conformation, a process driven by ATP. Often ATP hydrolysis occurs at the head regions of these proteins, while the "cargo" that they move along is held on the tail regions of motor proteins.

Protein Structure

This post will be mainly going over stuff that I've talked about before, but I'm including it for completeness and because I love HDs more than is really healthy. Anyhow...

Know the general formula of an amino acid.

An amino acid has an alpha-carbon joined to four different groups: a carboxyl group, an amino group, a hydrogen atom and a side chain, often referred to as R.

Be able to list the major groups of amino acids based on their side chains (e.g. basic, acidic, uncharged polar, nonpolar).

See my first post on amino acids.

Be able to describe the formation of a peptide bond.

See my earlier post on peptide bonds.

Understand that evolutionarily conserved proteins typically show amino acid sequence homology.

This dot point kind of speaks for itself. Proteins that have more or less retained their function over time and between related species tend to have similar or identical sequences.

Know the types of noncovalent and covalent interactions that may play a role in determining the conformation of a polypeptide.

See my earlier post on protein folding.

Be able to describe an α-helix and a β-sheet.
Be able to define the 1° (primary), 2° (secondary), 3° (tertiary) and 4° (quaternary) structure of a protein.

See my post on the levels of structure of a protein.

Be able to define a polypeptide domain.

A polypeptide domain is a region of a polypeptide that can fold independently into a stable structure. I *think* these domains might all have specific functions as well- for example, SH2 domains tend to bind to proteins that have phosphorylated tyrosine residues.

Translation- Part 2

This lecture doesn't have an outcomes slide, so I'm just going to expand on the summary as much as possible.

There are three potential translational frames in any mRNA; however, only one is used.

Translation of an mRNA can occur in one of three "reading frames." Let's say that we have an mRNA with bases 123456789. In the first reading frame, the first codon starts with base 1, so we have codons 123/456/789. In the second reading frame, the first codon starts with base 2, so we have codons 234/567. In the third reading frame, the first codon starts with base 3, so we have 345/678. There is no fourth reading frame because the end result is essentially the same as the first reading frame, minus the beginning codon. Although there are three different reading frames, only one will be used. The frame that will be used is usually one that produces a long chain without a stop codon.

Translation occurs in three steps: initiation, elongation and termination.

I will expand on these steps throughout the rest of this post...

Initiation of translation requires initiator Met-tRNAi in eukaryotes and fMet-tRNAi in prokaryotes.
When Met appears inside an open reading frame, Met-tRNA is used and this binds elongation co-factors.

As I may or may not have mentioned before, the codon that codes for methionine also codes for the start codon. Hence the initiating tRNA is always Met-tRNAi (the i stands for "initiator"). In prokaryotes, this initiating tRNA is slightly different- it's fMet-tRNAi, or N-formylmethionine tRNA, which is essentially Met-tRNAi bound to a formyl (aldehyde) group. The initiating tRNA is bound when the ribosome reaches a start codon. (This doesn't always happen- if I remember correctly sometimes the ribosome skips some start codons, resulting in "leaky scanning" and a different protein being produced. I'm not sure whether this happens in both eukaryotes and prokaryotes or only in eukaryotes, however.)

There are sequences on the mRNA to help guide the ribosome towards the correct start codon. In prokaryotes, the small subunit of the ribosome attaches to an initiation factor (IF), and then this complex binds to a special sequence called the Shine Dalgarno sequence, located upstream of the start codon. fMet-tRNAi then aligns with the start codon. The large subunit then binds, releasing IF. In eukaryotes, Met-tRNAi first binds to the small subunit, and then the loaded subunit scans along the mRNA to find the first AUG codon surrounded by a long consensus sequence known as the Kozac sequence. Initiation factors then detach, allowing the large subunit to bind.

Another important point to note is that the initiating Met-tRNAi is different to the Met-tRNA used in elongation. Met-tRNA has a slightly different shape, allowing it to bind more efficiently to elongation cofactors.

Elongation is a process using ATP to move the ribosome in the 5’ to 3’ direction, inserting tRNAs above codons and catalysing peptide bond formation between peptides.

During the process of elongation, the ribosome moves down the mRNA from 5' to 3', a codon at a time. At each position, an aminoacyl-tRNA enters the A site, the peptide in the P site joins onto the amino acid bound to the tRNA in the A site, and then the ribosome moves along. When the ribosome moves along, the tRNA originally in the A site goes into the P site, and the tRNA originally in the P site goes to the E site, where it leaves the ribosome. I've described this in more detail in an earlier post.

Termination occurs when the ribosome encounters a stop codon in the A-site, leading to a hydrolysis reaction, releasing the peptide from the final tRNA and causing dissociation of the ribosome from the mRNA. 

When the ribosome encounters a stop codon, release factors, rather than amino acids, bind. These release factors alter the activity of the peptidyl transferase (the enzyme that catalyses the addition of amino acids onto the chain). They also add water to the peptide, releasing it into the cytosol. The ribosome is then free to dissociate from the mRNA.

Translation- Part 1

Describe the genetic code.

The genetic code, in a nutshell, shows which codons code for which amino acids. (If you search it up on Google Images, you'll get plenty of tables.) It also shows which three codons are "stop" codons, which signal the end of translation. You'll never need to memorise the table- just knowing how to use it is enough.

Explain the history and experimental design associated with the discovery of the genetic code.

The genetic code was discovered through the work of several different scientists in the 1960s. In 1961, Nirenberg and Matthaei created artificial mRNA all made up of one kind of nucleotide- for example, they had one that only contained uracil, one that only contained adenine and so on. From this, they could create long strings of the same amino acid.

Later on, Khorana discovered a method that could make specific repeating mRNA strands. This helped scientists learn that the genetic code relied on series of three bases (codons). Nirenberg and Leder later used this knowledge to decipher all of the remaining codon sequences.

Explain the redundancy in the genetic code at the mRNA level.

There are 64 possible codons, as each codon is made up of 3 bases, and there are 4 different bases (4 x 4 x 4 = 64). However, there are only 20 different amino acids. A lot of amino acids can actually be coded for by multiple different codons: for example, UUU and UUC both code for phenylalanine. Hence there is redundancy in the genetic code at the mRNA level.

Explain the redundancy in the recognition of the genetic code by tRNAs using the wobble hypothesis.

As I mentioned, there are 64 codons. However, there are only around 40-50 tRNAs. How, then, can each codon find a corresponding tRNA to match up with?

The wobble hypothesis suggests that the third position of a codon is the least important, and inaccurate binding here may have less of an effect. For example, as I mentioned earlier, both UUU and UUC code for phenylalanine. Thus, it wouldn't be detrimental to the cell if phenylalyl-tRNA had an anticodon that could bind to both UUU and UUC.

One factor contributing to the wobble hypothesis is that tRNA often contain modified bases, such as inosine. Inosine can bind to U, A or C. Hence if phenylalyl-tRNA carried the anticodon 5'-IAA-3', it could bind to both UUU and UUC (remember, tRNA is antiparallel to mRNA, so the anticodon is going to look "backwards" compared to the codon).

Products of Transcription

Know the major classes of RNA molecules.

The three major types of RNA are mRNA, tRNA and rRNA. I've probably spoken about them before but I honestly reckon it'll be less effort for me to write it out again than go back and find a previous post about it. mRNA, or "messenger RNA," carry a transcript that will later be translated into a protein. tRNA carry amino acids to the ribosomes where mRNA is being translated. rRNA form parts of ribosomes, along with ribosomal proteins.

Know the differences between prokaryotic and eukaryotic mRNA.

I covered this in my previous post about the mechanism of transcription.

Know the structure and function of tRNA and function of aminoacyl-tRNA synthetases.

I covered this in a previous post about peptide bonds between amino acids. There is a different aminoacyl-tRNA synthetase for every amino acid, all named for the amino acid that they attach: for example, leucyl-tRNA synthetase attaches leucine to tRNA. As mentioned in the post that I just linked to, ATP is required for this process.

Know the differences between prokaryotic and eukaryotic ribosomes.

The main difference between prokaryotic and eukaryotic ribosomes is that eukaryotic ribosomes are larger.

Eukaryotic ribosomes are 80S, with a large 60S subunit and a small 40S subunit. The large subunit contains 5S, 5.8S and 28S rRNA whereas the small subunit contains 18S rRNA. (The S refers to the sedimentation coefficient or something. I'm not really sure how it's measured.)

Prokaryotic ribosomes, on the other hand, are 70S, with a large 50S subunit and a small 30S subunit. The large subunit contains 5S and 23S rRNA whereas the small subunit contains 16S rRNA.

Know the roles of ribosome binding sites in translation of mRNA to protein.

There are three binding sites in the ribosome: the A (aminoacyl) site, the P (peptidyl) site and the E (exit) site. The A site is where an aminoacyl-tRNA will initially enter the ribosome. After being added to the polypeptide chain, it will move across to the P site, and eventually along to the E site, where it exits the ribosome.

Know the definitions of polycistronic mRNA, polyribosome, mRNA cap, poly-A tail, codon, anticodon, amino acyl tRNA.

Polycistronic mRNA- mRNA that codes for multiple different proteins at the same time.
Polyribosome- Several ribosomes working to translate the same mRNA at the same time.
mRNA cap- A 7-methylguanosine "cap" that is attached to the 5' end of mRNA during eukaryotic mRNA processing. If I understood correctly, it's the place where the ribosomes bind onto the mRNA. The mRNA cap also provides some protection from early degradation.
Poly-A tail- A long series of A bases at the 3' end of mRNA. Also serves to protect against early degradation.
Codon- A series of three bases on the mRNA that code for either an amino acid or a stop codon.
Anticodon- A series of three bases on the tRNA that bind to a codon on the mRNA.
Aminoacyl-tRNA- tRNA with an amino acid attached at the 3' end.

Mechanism of Transcription

This lecture doesn't have a summary or an outcomes slide, so I'm just going to go through and talk about the stuff that looks important. (Given that it's me, though, everything looks important. I simply like those HDs far too much.)

The Central Dogma

I'm sure you probably know this by now- DNA gets transcribed to mRNA, mRNA gets translated into protein, yada yada yada.

Structure of RNA

See my earlier post on the properties of RNA.

Transcription

Transcription always takes place from 5' to 3', as with DNA replication. In fact you're probably going to be hearing "5' to 3'" so much that you'll be practically saying it in your sleep. Transcription is carried out by RNA polymerase, which does not require a primer (unlike DNA polymerase). It creates a strand of RNA that is complementary to the template strand of the DNA, and similar to the coding strand. In fact, the only difference between the coding strand and the initial transcript is that the coding strand has thymine while the initial RNA transcript has uracil.

RNA Polymerase

RNA polymerase is similar to DNA polymerase, but it doesn't require a primer and has a higher error rate. It uses the energy stored in ribonucleoside triphosphates in order to carry out its job.

In E. coli, RNA polymerase has five subunits- two alpha units, a beta unit, a beta prime unit and an omega unit. These five subunits are collectively known as the core enzyme of E. coli RNA polymerase. Another special subunit, sigma, is responsible for recognition of the promoter region of the DNA. It associates with the core enzyme to form a holoenzyme. After transcription initiation has taken place, the sigma subunit dissociates, leaving the core enzyme to continue elongation of the transcript.

Initiation and Termination Signals

Of course, for RNA polymerase to do its job properly, it has to know when to start and stop. Upstream of the gene, there are promoter regions. These are located at around 35 bases before and 10 bases before the start of the gene (a.k.a. -35 and -10. +1 is the start of transcription). The region between roughly -10 and -5 is known as the Pribnow box. These areas contain consensus sequences which are similar between genes and thus serve as a good guide for the RNA polymerase. If I remember correctly, promoters that have sequences more similar to the consensus sequence are considered to be "stronger" promoters than those that have sequences that contain more deviations from the consensus sequence.

Downstream of the gene, there are transcription termination signals. There are two main types of these. The first is a special DNA sequence in which there is an inverted repeat (i.e. the sequence is repeated but backwards the second time) followed by several thymine bases in a row. These are often translated to form "hairpin" structures in the mRNA. The second type of termination signal is a protein that binds to the DNA, physically blocking the movement of RNA polymerase.

Differences between Prokaryotic and Eukaryotic mRNA

There are several differences between prokaryotic and eukaryotic mRNA. Firstly, prokaryotic mRNA is often polycistronic- that is, it codes for multiple proteins at the same time- whereas eukaryotic mRNA is often monocistronic (one protein at a time). Prokaryotic mRNA may also have intercistronic regions, or spacers, located between the coding regions. Another important difference between the two types of mRNA is that prokaryotic mRNA is not processed, whereas eukaryotic mRNA is. Eukaryotic mRNA has a 7-methylguanosine cap added on its 5' end and a poly-A tail located on its 3' end. Also, eukaryotic mRNA has non-coding sequences called introns (prokaryotic mRNA does not have these) that are spliced out during mRNA processing.

Mutation and Repair of DNA

Explain mutation of DNA.

Mutation is defined as a heritable change in the DNA. It can take place on a smaller scale (e.g. a change of bases) or a larger scale (e.g. chromosomes fusing together).

Describe the processes that result in the mutation of DNA.

There are several different ways in which mutations can be acquired. Some arise from errors when the DNA is replicated. This is a very rare occurrence- DNA replication and the DNA proofreading processes are so good that the error rate is only 1 in 10^9 bases. Exposure to certain environmental factors, such as radiation, certain chemicals and infectious agents, may also cause damage to the DNA.

Describe the consequences of depurination, deamination, thymine dimer formation and double stranded breaks on DNA replication.

Depurination is the loss of a base from the DNA backbone. During replication, this base will simply be skipped.

Deamination is the loss of amine groups from bases. This can cause unnatural bases in the DNA, which may pair up with different bases during DNA replication. For example, when cytosine is deaminated, it forms uracil, which will pair up with adenine during DNA replication.

Thymine dimers can occur when DNA is exposed to UV light. A thymine dimer is when two adjacent thymine residues bind to each other rather than to their complementary bases on the other strand.

Double-stranded breaks are basically when both strands are broken. There are also single-stranded breaks in which one strand is broken. One cause is exposure to gamma or X-rays, which either produce free electrons or generate hydroxide radicals, both of which can attack the DNA backbone.

Understand how transposable DNA elements and infectious agents introduce mutations into DNA.

Transposable DNA elements and infectious agents can integrate themselves into DNA, which in turn can disrupt the function of whatever part of the DNA they happen to find themselves in. For example, if a transposable element is moved into the middle of a region coding for a gene, the function of this gene will be disrupted. Transposons essentially enter via a cut-and-paste kind of method: transposases (which are encoded by the transposons themselves) "cut" out the transposon from the original DNA and then "paste" it somewhere along the host DNA strand. Once in the new DNA strand, it can continue to replicate along with the DNA.

Explain the two mechanisms for DNA repair: MisMatch repair system and homologous recombination.

As mentioned above, DNA has several proofreading and repair mechanisms that are partially responsible for the low rate of mutations.

Mismatch repair is a relatively simple process. Firstly, mispaired bases are recognised by repair proteins. This may be due to the change in thickness of the strand at that point- as alluded to in an earlier post, two purines or two pyrimidines pairing together may result in the DNA being too thin or thick at that point. The wrong base is removed by nucleases. The correct base is then added in and the gap in the backbone is sealed with DNA ligase.

Homologous recombination is relatively complex and is used to repair double-stranded breaks in the DNA. It is called "homologous" because extensive regions of similarity are required for it to work. Often it takes place right after the DNA has replicated. The first step of homologous recombination is to remove some nucleotides from the 5' to 3' direction, leaving 3' overhangs. These overhangs can then move into a chromosome with the same sequence (as I said, it generally takes place right after DNA replication, when such chromosomes are available), where DNA polymerase can add more nucleotides to them. (The region where the overhangs "cross over" into the other chromosome is known as the Holliday junction.) When enough DNA has been synthesised, the chromosomes can be resolved by strand cutting. A similar process is also used for crossing over of chromosomes during prophase I of meiosis.

DNA Replication

Define the terms describing DNA replication: semiconservative, origin, bidirectional, replication fork, Okazaki fragment.

Semiconservative: When DNA replicates, each daughter molecule of DNA has one strand from the original DNA and one new strand. This means that it is semiconservative (as opposed to conservative in which the entire daughter molecule would be made of original DNA).
Origin: The place at which DNA replication takes place. Eukaryotic chromosomes often have multiple origins.
Bidirectional: Proceeding in two directions at once.
Replication fork: The area where the DNA is currently being replicated. The two DNA strands are separated in this area with DNA replication taking place on both.
Okazaki fragment: Fragments of DNA that are formed on the lagging strand. (I'll explain this in a bit.)

Understand the mechanism of leading and lagging strand replication and role of the RNA primer.

After the DNA has been opened up to reveal the replication fork, DNA synthesis takes place on both strands. DNA is antiparallel so the two strands are running in opposite directions to each other, and thus synthesis must take place in opposite directions as well.

Synthesis, however, must run from 5' to 3'. On one strand, this works quite well, and the DNA can be synthesised continuously. This strand is known as the leading strand. On the other strand, otherwise known as the lagging strand, the DNA is synthesised in little fragments at a time as the helix is opened up. These fragments are known as Okazaki fragments.

About primers- DNA polymerase can only join nucleotides to already existing nucleotides. The cell gets around this by using RNA primers, as RNA doesn't need already existing nucleotides to form a new strand. An RNA primer is placed at the start of every leading strand and at the start of every Okazaki fragment on the lagging strand. In the case of the lagging strand, most of these RNA primers are removed by RNAse H before DNA polymerase "overwrites" them with DNA.

Understand the functions of the proteins at the DNA replication fork.

There are several different proteins at the DNA replication fork. They cooperate to form what is known as the "replication machine." One of these is helicase, which unzips the double helix. (There's a really bad joke that goes "If I was an enzyme, I'd be helicase so that I can unzip you.") The sliding clamp is a protein that holds DNA Polymerase in place during replication, whereas the clamp loader assembles the clamp onto the DNA using energy from ATP. There are also single strand DNA-binding proteins that stabilise the unwound strands of DNA, preventing them from "snapping back together."

List major DNA polymerases of prokaryotes and eukaryotes and their functions.

There are three main types of DNA polymerase in prokaryotes such as E. coli. These are very imaginatively named DNA polymerase I, II and III. DNA polymerase I has functions in DNA repair as well as the maturation of Okazaki fragments (i.e. the removal of the RNA primer and the "stitching up" of Okazaki fragments to form a continuous strand). DNA polymerase II also plays roles in DNA repair. DNA polymerase III is the main DNA replication enzyme in prokaryotes, and it has a much higher affinity for deoxynucleotides than the other two types of DNA polymerase and is therefore able to add nucleotides much more rapidly.

There are five main types of DNA polymerase in eukaryotes. They are similar to those in prokaryotes but have some more protein components. Also, instead of being named I, II, III etc., they are named after letters of the Greek alphabet. Alpha DNA polymerase elongates primers, beta DNA polymerase repairs DNA, gamma DNA polymerase replicates mitochondrial DNA, delta DNA polymerase plays roles in the synthesis of the lagging strand and epsilon DNA polymerase plays roles in the synthesis of the leading strand. Please do note, however, that the roles of delta and epsilon DNA polymerases are still controversial.

DNA Structure

Relatively simple topic for this post- should be my last one tonight.

List hypotheses on the origin of life on earth.

There are many different hypotheses for the origin of life on Earth, but only four were covered in the course.

#1: Organic chemical synthesis in a reducing atmosphere. It was thought that the Earth originally had a reducing atmosphere, in which hydrogen and methane gases were abundant. The significance of this is that it has also been found that hydrogen, methane and ammonia can form a "prebiotic soup" made of amino acids and nucleotides when exposed to water and electrical discharges. However, the current consensus is that the primitive atmosphere was not reducing. It is also unclear how membrane-enclosed cells could have formed.

#2: Carriage by meteorites/comets. Part of the evidence behind this hypothesis is the abundance of organic compounds found in space. The issue with this hypothesis, however, is that it simply changes the question to "How did life originate in space?"

#3: Synthesis on metal sulfides in deep sea vents. Apparently vents are sites of abundant biological activity that can form prebiotic soups similar to those mentioned in #1 (well, that's my understanding anyway). The prebiotic soup here can self-organise on a metal sulfide surface. Once again, however, the question of how membrane-enclosed cells are formed is raised.

#4: RNA World. This hypothesis suggests that RNA may have been the first living, self-replicating entity. Some RNA is capable of storing information and/or catalysing reactions. Yet again, the question is raised of how membrane-enclosed cells are formed.

Know the main events which led to the discovery of DNA.

Watson and Crick are the two scientists mostly credited for the discovery of the structure of DNA, but there were also several others who contributed towards their discovery. Watson and Crick worked together in the same laboratory. Watson was first shown an X-ray diffraction image of DNA by Maurice Wilkins at a conference. Later on, Rosalind Franklin produced an even better image. This data allowed Watson and Crick to propose their first model of DNA, which was a three-stranded model.

This model, as I'm sure you know, was found to be incorrect. It was also inconsistent with Franklin's data, as she pointed out to the pair. The next year, Watson and Crick were visited by Edwin Chargaff, who showed them his data about the ratio of A/T and G/C in DNA, thus providing another small piece of the puzzle. Yet more data was obtained from later X-ray diffraction data taken by Wilkins.

Eventually, in 1953, Watson and Crick used all of the information that they had gathered to produce a double-stranded helical model of DNA. Apparently, after they had their "Eureka!" moment, they went to the local pub to announce that they had found out the secret of life. (I wonder how many weird looks they got for that?) Watson, Crick and Wilkins all obtained the Nobel Prize. Unfortunately, Franklin did not receive the Nobel Prize, as she had died of ovarian cancer before the prize was awarded, and you cannot receive a Nobel Prize posthumously.

Be able to describe the main features of DNA.

DNA is made up of two antiparallel strands of nucleotides. Each nucleotide is made up of deoxyribose sugar, which is bound to a base at the 1' carbon and a phosphate group at the 5' carbon. The bases on the two strands are hydrogen bonded to each other, with adenine pairing to thymine and cytosine pairing with guanine. The two strands are wound around into a right-handed double helical structure.

Understand how the genetic code works.

Messenger RNA has series of "codons." Each codon is three bases long and codes for one amino acid. The genetic code is degenerate, which means that several different codons can all code for the same amino acid. There are also three codons that are "stop" codons- that is, instead of coding for an amino acid, they bind release factors which cause the ribosome to release the polypeptide.

Know what is meant by “the central dogma.”

The central dogma is simply the process in which DNA is transcribed into mRNA, which is then translated into protein.

Know the terminology for bases, nucleotides, nucleosides, deoxy, ribo.

There are several different bases present in RNA and DNA. These include adenine, thymine, cytosine, guanine and uracil. (There are other modified bases, but they're not important to us at this stage.)

When these bases are bound to a sugar (ribose or deoxyribose), they form a nucleoside. These nucleosides are called adenosine, thymidine, cytidine, guanosine and uridine. (I'm sure you can guess which nucleoside contains which base.)

A nucleotide is a nucleoside bound to one or more phosphates. For naming, you take the nucleoside name and then "monophosphate" or "diphosphate" or whatever-phosphate. For example, adenosine with three phosphate groups bound is adenosine triphosphate- otherwise known as ATP, an energy carrier in the cell.

If you want to be really precise, you can also add deoxy- or ribo- prefixes to nucleoside and nucleotide names in order to make it clear whether the sugar is deoxyribose or ribose, respectively.

Be able to define the terms antiparallel, complementary base pairing, coding strand, codon, right-handed helix, major groove.

Antiparallel- The two strands are parallel to each other, but run in opposite directions.
Complementary base pairing- Bases bind to complementary bases: adenine always pairs with thymine (in DNA) or uracil (in RNA) and cytosine always pairs with guanine.
Coding strand- The strand opposite to the template strand. (The template strand is the one that is copied off during the process of transcription.)
Codon- A series of three bases on the mRNA. Each codon codes for an amino acid (except for the three stop codons, which cause release factors to bind). Multiple codons may code for the same amino acid.
Right-handed helix- A clockwise helical structure.
Major groove- A relatively large groove in the side of the DNA helix.

Cell Cycle Control

I have spoken about the cell cycle in previous posts. Now it's time to explain how the cell cycle is controlled!

Know the meanings and importance of the key words.

Homeostasis- Maintenance of a stable condition. For example, in the body, temperature and pH are maintained at a certain level.
Cell cycle checkpoints- Points in the cell cycle where the cell "checks" to make sure that everything is okay before proceeding on to the next stage. Once the cell passes a checkpoint, it cannot go back.
Cyclin- A protein that binds to a cyclin-dependent kinase (Cdk) in order to regulate their function. There are different types of cyclins that are produced at different stages of the cell cycle.
Cyclin dependent kinase (Cdk)- A kinase (i.e. an enzyme that adds phosphate groups to proteins) that needs to be bound to a cyclin in order to function. Cdk levels remain constant throughout the cell cycle, but the proteins that they phosphorylate differ according to which cyclin is bound to the Cdk.
p53, a tumour suppressor- p53 is essentially a protein that either tells the cell not to replicate the DNA if there are some mismatches or to commit suicide if the DNA is severely damaged. (What a bully, picking on damaged cells like that.) It works by recognising DNA mismatches and then activating other proteins. In the case of a smaller amounts of damage, p53 activates p21, which stops Cdks from phosphorylating their target proteins, which in turn prevents entry into the S phase until the DNA is repaired. In the case of larger amounts of damage, p53 activates genes that code for apoptosis (see my previous post for more information on apoptosis).

Have an understanding of the cell cycle phases and the checkpoints where control is exerted.

I think I've already gone over the cell cycle before, but now seems like a good time to go over it again.

The first stage of the cell cycle is the G1 phase, in which cells do most of their growing and stuff. Some cells leave the cell cycle at this point and enter the G0 phase, where they remain pretty much stable for the rest of their lives. (Neurons and skeletal muscle cells are prime examples.) Cells that are destined to divide pass through the first checkpoint: the G1/S checkpoint, also known as Start. At this point, the cell ensures that the DNA is intact and that other environmental conditions are favourable for division. If everything is good, the cell proceeds on to the S phase.

In the S phase, the cell replicates its DNA. Once that is finished, the cell is said to be in the G2 phase, during which it undergoes a little more growth. At the end of this stage, there is another checkpoint: the G2/M checkpoint. At this stage, the cell checks to ensure that DNA replication has been completed properly.

Finally, the cell undergoes the M phase, otherwise known as the mitotic phase. I have written about the stages of the mitotic phase in an earlier post. Between metaphase and anaphase, there is another checkpoint, known as the spindle assembly checkpoint. At this stage, the cell checks to make sure that the spindle fibres have formed properly and are attached to the chromosomes.

Understand that cyclin levels oscillate with cycle phase.

As I mentioned in the definitions, cyclins are proteins that bind to and activate cyclin-dependent kinases (Cdks). Cyclins also determine which proteins a Cdk will phosphorylate. If I remember correctly, often a cyclin bound to a Cdk will activate the production of the next cyclin needed. For example, G1/S-cyclins needed to pass through Start will stimulate the production of S-cyclins, which are needed for the events that take place during DNA replication.

Realise that Cdk activity is controlled by cyclin binding, phosphorylation and inhibitors.

As also mentioned in the definitions, Cdks require cyclins to activate them (hence they are cyclin dependent kinases). Like all kinases, Cdks work by adding phosphate groups to their target proteins. Cdks themselves can also be activated or inactivated by the addition of phosphate groups.

Cell Cycle and Apoptosis

Realise the importance of the cell cycle.

The cell cycle basically involves the processes of growth and replication. I'm sure you can agree that those processes are pretty important without me having to explain why. Also growth and replication have to be coordinated so that you don't end up with a massive cell that hasn't divided yet or lots of tiny cells with not enough stuff in them.

Understand the key steps in mitosis.

See one of my earlier posts on cell structure and mitosis.

Know chromosome structure and how chromosomes behave during the eukaryotic cell cycle.

I described chromosome structure in my first post on eukaryotic gene regulation. In the G1 phase of the cell cycle (i.e. the growth phase before DNA replication), cells have the full complement of chromosomes. In humans, this is 46 chromosomes (23 pairs). Each chromosome has one DNA double helix. In the S phase, the DNA replicates (aside from at the centromere), and so each chromosome has two DNA double helices joined together at the centromere. Each double helix is called a sister chromatid. During cell division, the centromere also replicates and the chromatids are pulled apart.

Remember, one centromere = one chromosome. Despite post S-phase chromosomes having two chromatids, there are still only 46 chromosomes until the centromeres are replicated and the chromosomes are pulled apart during mitosis.

Understand the key steps in meiosis.
Know the differences between mitosis & meiosis.

Both of these are covered in a previous post on reproduction.

Understand the need for, and process of, apoptosis.

Apoptosis is the controlled death of cells. Apoptosis has several different functions. During embryonic development, for example, cells that are needed for earlier development, but not for later development, may undergo apoptosis. A more specific example of this is the webbing between our fingers. As embryos we have far more webbing until some of the cells in the webbing die off. Other reasons for apoptosis may be to kill off diseased cells.

Caspases are enzymes that drive the process of apoptosis. Before they are signalled, they exist in a harmless, inactive state known as a zymogen. After receiving a signal, which can be extracellular or intracellular (I did read up a bit more on these two pathways, but I don't remember them and I think they're beyond the scope of this unit anyway), initiator caspases are cleaved. Initiator caspases then cleave executioner caspases, which in turn cleave other target proteins in the cell so that apoptosis can take place.

At the beginning of apoptosis, executioner caspases cleave a protein that normally holds DNA endonuclease in the cytoplasm. When this protein is cleaved, DNA endonuclease enters the nucleus and eats up the DNA. Executioner caspases then activate an actin-cleaving protein, resulting in degradation of the cytoskeleton and a loss of cell shape. The cell breaks up into small fragments called apoptotic bodies. These apoptotic bodies are later phagocytosed (eaten up) by other cells in the body. Om nom nom. (Sorry, just had to put that there.)

Eukaryotic Gene Expression- Part 2

Know the meanings and importance of the key words

(Again, these are my own half-assed definitions, not proper textbook definitions, so they might not be 100% accurate.)

Transcription factor: Proteins that bind to the DNA to promote transcription of genes.
Promoter: The area that directs the binding of RNA polymerase II. Promoters are located close to the start site. There are also enhancer regions, which may also help regulate genes, but these are often located far away from the start of the gene.
Regulatory sequences: Binding sites for regulatory proteins. Can be located pretty much anywhere, even within introns.
Half-life: The time that it takes for half of a particular type of molecule to be degraded.
mRNA degradation: The degradation of mRNA... This generally starts with shortening of the poly-A tail by an exonuclease. When the tail is short enough, degradation speeds up.
UTR (untranslated region): Parts of mRNA that are not translated.

Have an understanding of the mechanisms of control of gene expression from transcriptional to translational.
Understand the different facets of transcriptional control.
Realise that transcription factors bind sequentially.
Understand intron-exon splicing.

Transcription

Just like for prokaryotes, the main point of control is at the transcriptional level. This is regulated by cis-acting control elements, which are motifs and sequences along the DNA itself. These include promoter, enhancer and repressor regions- see my definition of "promoter" above.

Around 25 base pairs from the starting site, there is a sequence known as the "TATA box" as it contains the TATA sequence. This binds TATA-binding protein, or TBP. TBP is a part of transcription factor for RNA polymerase II, subunit D, otherwise known as TFIID. TFIID is one of several transcription factors that bind near the TATA box. They all bind in a specific order, though I don't think knowledge of the order is necessary at this stage. Sometimes other proteins might bind to enhancers, which are often located far away from the start site. Proteins at both the TATA box and at the enhancer can be joined together by other proteins known as mediators.

When everything is in place, RNA polymerase can bind to the DNA. RNA polymerase II, which creates mRNA transcripts, has a tail known as the CTD, or C-terminal domain. For transcription to begin, the CTD must first be phosphorylated.

(Oh, and by the way, there are other RNA polymerases. I'll just include them here for completeness. RNA polymerase I is used to synthesise most types of rRNA, including 5.8S, 18S and 28S rRNA. RNA polymerase III is used to synthesise 5S rRNAs as well as most other types of RNA in the cell.)

Pre-mRNA Processing

Once the gene has been transcribed, it is time for pre-mRNA processing. Processing at this stage is also vital to the function of the proteins that are being produced. Processing includes the splicing out of introns (non-coding regions) and the splicing together of exons (the coding regions). Exons can also be spliced in different orders, resulting in many different proteins that can be produced from the same gene depending on how it is spliced. Processing also includes the addition of a 7-methylguanosine cap on the 5' end and a poly-A tail on the 3' end, both of which contribute towards the stability of the mRNA.

The enzymes and other factors required for capping and splicing mRNA are located on the C-terminal domain of RNA polymerase. The addition of the poly-A tail is catalysed by poly-A polymerase.

Splicing is quite complex, and so I won't go into a lot of details now (mainly because I don't know a lot of details at this stage :P). The pre-mRNA has special splicing sequences that indicate where splicing is to occur, and these are recognised by the "spliceosome"- a complex of proteins that catalyse splicing. Many of these proteins are snRNPs, or small nuclear ribonucleoproteins.

mRNA Transport and Localisation

mRNA is often translated in places where its products will eventually be needed, as this is often more efficient. Also, damaged mRNA is prevented from leaving the nucleus at all by being taken to exosomes to be degraded by exonucleases.

There are several ways through which mRNA can be localised to a specific location. Firstly, mRNA can be transported via cytoskeletal proteins. Secondly, mRNA can diffuse through the cytosol to later become trapped in certain locations. Finally, mRNA may diffuse through the cell, progressively degrading everywhere except for in certain locations where protective factors are present.

Translation

Most translational control occurs at the initiation of translation. The 5' cap is required for the binding of the small ribosomal subunit. Additionally, certain eukaryotic initiation factors have to bind to the 5' and 3' ends to allow translation to occur, thereby ensuring that translation only occurs on mRNA that are intact.

Degradation

The half-lives of eukaryotic mRNA can differ from only a few minutes to many hours. They are determined by specific sequences in the mRNA. As I mentioned before, the poly-A tail is shortened before the rest of the mRNA is degraded. Proteins that catalyse these processes compete with translational proteins that bind to the same region. Hence translation and degradation will not occur at the same time.

Eukaryotic Gene Regulation- Part 1

In the past few posts, I covered regulation of genes in prokaryotes, using the tryptophan and lactose operons as examples. Now I'm going to talk about eukaryotic gene regulation, which will be a bit more complex as eukaryotes are more complex.

Know the meanings and importance of the key words.

(These are just my shitty definitions, not textbook definitions, so take them with a pinch of salt.)

Somatic, germ line- Germ line cells are cells that produce the gametes (eggs and sperm). Somatic cells are all of the other cells in the body.
Zygote- The cell that forms when the egg and sperm combine.
Chromatin- Complexes formed from DNA and protein. Can condense to form chromosomes.
Chromosome- Condensed chromatin. There are 46 chromosomes in a normal human cell.
Differentiation- Cells developing special features to transform from a totipotent (i.e. can become anything) stem cell to a particular mature cell in the body.
Development- The process in which cells grow and develop...? During this process, they become differentiated.
Chromatin remodelling- The modification of the chromatin in order to make the DNA more or less accessible to RNA polymerase. For example, DNA can become more or less condensed.
Histone modification- The modification of amino acids in the histones of chromatin. This may make the DNA more or less accessible.
Acetyltransferase- Enzymes that add acetyl groups to lysine residues in histones. This weakens the interaction of histones with the DNA, thereby making the DNA more accessible to RNA polymerase. This can be reversed by deacetylases.
DNA methylation- The addition of methyl groups to the DNA. Methylation of genes may cause silencing.
Morphogen- A signalling molecule that helps guide the process of differentiation.

Have a general understanding of the process of differentiation.

Differentiation, as I mentioned, is the process in which an undifferentiated cell undergoes changes to become a particular differentiated cell type. The first totipotent stem cells produced from the zygote dividing will eventually proliferate and divide to produce neurons, bone cells, blood cells etc. Remember, proliferation must proceed differentiation- you can't have one cell differentiate into 200+ types without the one cell dividing first. Differentiated cells usually have a distinct morphology (appearance) that helps us to identify them.

Differentiation is accomplished partly by gene regulation. This, in turn, may be influenced by transcription factors, which can be either general or cell/tissue specific. Contact with hormones or morphogens may also influence gene expression. Other enzymes that modify chromatin may also influence differentiation through influencing the ability of genes to be expressed.

Realise the complexity of chromosome structure & its role in gene expression.

Chromosomes are not simply long strands of DNA and protein- they are coiled a lot so that all of that information can fit inside the tiny nucleus of the cell. Some of the most prominent proteins in chromosomes are histone proteins. Eight of these fit together to form a histone octamer, around which the DNA winds. The DNA plus the histone octamer is known as a nucleosome. Nucleosomes are joined by small bits of DNA called "linker DNA." Nucleosomes, in turn, can form a spiral known as a solenoid, which is stabilised by another histone protein. Solenoids can condense even further to eventually form the banding patterns that can be seen on chromosomes. All of this coiling allows the DNA to be packed tightly without it getting so tangled up that transcription factors cannot bind.

Positive Regulation of the Lactose Operon

I covered negative regulation of the lactose operon in my previous post... now it's time to talk about positive regulation!

List the components important in positive regulation of the Lac operon.

The main components involved in positive regulation are the Lac promoter, the activator and cAMP (cyclic AMP). The promoter on the Lac operon is a weak promoter, so under normal conditions, RNA polymerase will not recognise it well. The activator, CAP, binds to cyclic AMP to form a CAP-cAMP dimer. This dimer binds to a region of DNA near the promoter, allowing RNA polymerase to bind to the DNA more readily. This, in turn, allows for transcription of the genes.

Describe the positive regulation of the Lac operon.

The main thing to understand here is that cAMP levels are inversely proportional to glucose levels. As glucose levels rise, cAMP levels drop, and when glucose levels drop, cAMP levels rise. As the function of the Lac operon is to allow the cell to metabolise lactose in the absence of glucose, you might expect that low glucose levels, and therefore high cAMP levels, will allow for transcription of the gene. This is exactly what happens: as I mentioned above, cAMP can bind to the activator protein CAP, forming a CAP-cAMP dimer that can bind near the promoter, inducing the transcription of the genes.

List 3 regulatory DNA-binding proteins in bacteria.

Easy: I've already mentioned three in this post and in my previous posts about gene regulation. TrpR is a repressor that binds to the tryptophan operon, LacI is a repressor that binds to the lactose operon and CAP is an activator that binds to the lactose operon.

Understand how proteins can bind to specific regions of DNA, and the roles of some conformational changes caused by this binding.

Proteins can bind to the outside of DNA, particularly in the major groove of the double helix. Amino acids on the protein can form hydrogen bonds with the sides of bases in the double helix. (Proteins cannot actually insert themselves into the double helix.) A common DNA-binding motif is the helix-turn-helix motif. One of the helices is called the recognition helix as it helps the protein to recognise the major groove of the DNA. Generally these proteins exist as dimers (proteins with two subunits), with the two recognition helices separated by exactly one turn of the DNA helix.

Binding of proteins to the DNA can cause conformational changes to the DNA, which in turn can assist or prevent the binding of RNA polymerase. For example, the CAP-cAMP dimer bends the DNA by 90 degrees, facilitating the binding of RNA polymerase to the DNA. Also, as mentioned in my previous post, other proteins can create loops that prevent the binding of RNA polymerase to the DNA.

The Lactose Operon

Draw a graph to illustrate usage of glucose and lactose in E. coli.

Okay, you probably know the drill by now. I'm not going to draw anything, because I'm lazy, and besides there are plenty of better graphs on Google Images. Just google "growth of E coli glucose lactose" and you will find several that show what I'm about to describe.

The growth of E. coli in glucose and lactose occurs in two stages, and thus is known as "diauxic growth." Firstly, E. coli consumes glucose, as that is its preferred source of energy. Once the glucose has been consumed, E. coli begins to transcribe the genes needed for metabolism of lactose. While this is occurring, growth temporarily stops or slows down considerably. Once the genes have been transcribed, E. coli begins consuming lactose, and its growth starts again.

List the enzymes of the lactose operon and their functions.

The lactose operon produces two main enzymes: beta-galactosidase and lactose permease. Lactose permease transports lactose into the cell, whereas beta-galactosidase hydrolyses lactose into glucose and galactose. Beta-galactosidase also has a side pathway in which lactose is converted to allolactose. This is the inducer for negative regulation, as we shall see in a bit. The lactose operon also has a section coding for transacetylase, the function of which is still unclear.

Describe the lactose operon and its negative regulation.

The lactose operon consists of six regions: IPOZYA. The I part codes for the repressor protein. P and O are promoter and operator, respectively- see my previous post for definitions of these terms. Z codes for beta-galactosidase, Y codes for lactose permease and A codes for transacetylase.

The negative regulation of the lactose operon involves the repressor coded by the I region of the operon. (Remember, negative regulation always involves a repressor.) This time, instead of a co-repressor helping the repressor bind to the DNA, there is an inducer that prevents the repressor from binding to the DNA. In this case, the inducer is allolactose. When allolactose binds to the repressor, the repressor does not bind to the DNA, allowing transcription to take place. (Glucose also needs to be either not present or present only in low levels, but I'll cover that in my next post when I talk about positive regulation.)

I'm going to talk a little bit more about the repressor protein itself because it's interesting enough to have slides devoted to it. The lac repressor is a tetramer- that is, it's made up of four polypeptide chains bound together via mainly noncovalent bonds. It has two identical binding sites that can bind onto the DNA. The lac operator actually has three binding sites: O1 (the main site), which is between P and Z, O2, which is after Z, and O3, which is before P. The repressor binds to O1 and either O2 or O3. A loop is formed, which contains the sites that are usually recognised by RNA polymerase. Hence, binding of the repressor to form a DNA loop prevents binding by RNA polymerase and therefore also prevents transcription.

Another interesting point to make about negative regulation of the lactose operon is the "chicken and egg" problem. Allolactose is required to remove the repressor, allowing transcription of the mRNA for beta-galactosidase and lactose permease. However, beta-galactosidase is required for the production of allolactose in the first place! How do cells get around this? Well, in my previous post I mentioned that prokaryotic genes generally cannot be turned off, but rather have their activity reduced to a low basal level. Hence there is always a little beta-galactosidase around to convert lactose to allolactose.

Explain the terms inducer, inducible, on-off regulation and diauxic growth.

An inducer, as I've mentioned before, is a protein that binds to a repressor in order to prevent the repressor from binding to DNA, thus allowing transcription of the gene. An inducible protein is a protein produced in this manner.

On-off regulation is the switching on or off of genes due to the presence or absence of repressors or activators.

Diauxic growth is growth in two stages, such as the growth of E. coli in the presence of glucose and lactose.

Regulation of Gene Activity in Prokaryotes

List differences between gene regulation in prokaryotes and eukaryotes.

In eukaryotes, genes can be switched "on" and "off." In prokaryotes, however, genes are rarely switched off completely- rather, they will transcribe at a low, basal level.

Another difference between prokaryotes and eukaryotes is that in prokaryotes, transcription and translation occur at the same time whereas in eukaryotes they occur separately. Also, prokaryotes have polycistronic mRNA- mRNA that code for multiple proteins- whereas eukaryotes only have monocistronic mRNA that code for one protein at a time. These have further implications in gene regulation, as we shall see.

Understand the effect of and reasons for gene regulation.

The reasons for gene regulation are simple: to make sure that cells have the proteins they need when they need them and don't have an excess of proteins that they don't need. Gene regulation helps to achieve this.

List four ways that a cell can control the proteins it makes.

Transcriptional control- controlling when and how much of a gene is transcribed.
RNA processing control- controlling the splicing and other modifications to the mRNA. I *think* this probably happens more in eukaryotes as transcription and translation do not occur simultaneously.
Translational control- controlling which mRNA get translated.
Post-translational control- controlling the activity of a protein that has already been translated. This can be done by adding phosphate groups etc.

Describe the basic principles of coordinate regulation, catabolic vs. anabolic pathways, and positive vs. negative regulation.

Coordinate regulation refers to the simultaneous transcription of all of the proteins needed for a particular pathway. For example, let's say that degradation of a particular molecule requires enzymes A, B and C. Since all of them are needed or not needed at the same time, you are likely to find polycistronic mRNA in prokaryotic cells that encodes all three enzymes.

Catabolic pathways are those in which larger molecules are broken down into smaller ones, and anabolic pathways are those in which smaller molecules are combined to form larger ones. In catabolic pathways, often the availability of the larger molecule to be degraded determines whether or not the enzymes needed are synthesised. In anabolic pathways, the reverse happens: the final product tends to regulate the synthesis of the enzymes. I think the final product here also tends to decrease the synthesis of anabolic enzymes- after all if you already have a lot of final product, you probably don't need too much more.

Positive and negative regulation depends on whether activators or repressors are used. In positive regulation, an activator binds to the DNA, increasing the transcription of the gene. This may be by making the promoter site more susceptible to binding by DNA polymerase. In negative regulation, a repressor binds to the DNA, decreasing the transcription of the gene.

Describe the tryptophan operon of E. coli and its negative regulation.

The tryptophan operon codes for the synthesis of enzymes that produce tryptophan. This is an example of an anabolic pathway, since tryptophan is being produced by smaller molecules. Like many anabolic pathways, the end product (tryptophan) controls the synthesis of enzymes- in this case, it decreases the synthesis of enzymes so as to prevent over-production of tryptophan.

The regulation of the tryptophan operon is also an example of negative regulation. The tryptophan repressor, TrpR, can bind to the DNA in order to stop the transcription of enzymes that produce tryptophan. TrpR binds to the DNA when tryptophan is bound to it. In this case, tryptophan is known as a corepressor, as it causes the repressor to bind to the DNA. (There are also inducers, which stop repressors from binding to DNA. I'll talk about one of these in a later post.)

Define the terms operon, promoter, operator, repressor and polycistronic mRNA.

I'm not really sure how to define "operon," but it seems to be a section of DNA that has a promoter, an operator and several genes that usually code for enzymes in the same pathway. Operons tend to produce polycistronic mRNA- mRNA that can code for multiple different proteins.

A promoter is the place where the RNA polymerase binds to during the transcription of mRNA.

An operator is the place where repressors or activators bind in order to prevent or induce transcription of a gene.

A repressor is a molecule that prevents the transcription of DNA when it is bound.

Copying DNA and RNA in vitro

Know the components of a PCR.
Know the steps in a PCR cycle.
Be able to draw a diagram illustrating the binding of a PCR primer pair to a complementary double-stranded template and extension of the primers by Taq polymerase. 

A PCR, or Polymerase Chain Reaction, is a method used for creating many copies of a DNA or RNA of interest. It involves firstly denaturing the DNA, attaching primers to the separated strands and completing synthesis using DNA polymerase.

DNA is denatured by increasing the heat. 20-30 base long primers, which have been specifically designed (some knowledge of the sequence is required to carry out PCR) anneal to the DNA as the temperature is lowered and flank the region to be amplified. DNA polymerase, which is taken from thermophilic bacteria (i.e. bacteria that live in hot regions) is used to replicate the DNA as it will not denature at the high temperatures.

This process can be carried out in a thermocycler- a machine that increases and decreases the temperature at each stage of the cycle.

Know the properties of reverse transcriptase.

Reverse transcriptase is an enzyme that synthesises DNA from an RNA strand. Before it can do this, a poly-T primer binds to the poly-A tail of mRNA, as reverse transcriptase requires a primer. Reverse transcriptase then makes a copy of the RNA strand using deoxyribonucleotides. Next, RNase H degrades most of the RNA strand. Finally, DNA polymerase comes in and re-synthesises that strand using DNA. The DNA strand that is produced is known as cDNA, or complementary DNA, as it is a complement of the mRNA strand. This cDNA can be replicated many times in a process similar to PCR, but in this case it is known as RT-PCR: Reverse Transcriptase PCR.

Know the differences between a PCR and an RT-PCR and when each is used.

As mentioned above, PCR replicates parts of DNA strands, whereas RT-PCR replicates cDNA, or DNA complementary to mRNA strands. The main difference here is that cDNA does not have introns and may have sections shuffled around due to alternative splicing. PCR can be used to examine and replicate genes that are present in the DNA regardless or whether they are expressed or not, whereas RT-PCR can be used to study the expression of genes.

Hybridisation Techniques

Be able to describe the conditions under which double-stranded nucleic acid molecules are denatured and renatured.

As I believe I've mentioned in a previous post (though I can't be bothered trying to find out which one right now), high temperatures and high pH both cause DNA to become denatured. High temperatures denature DNA by breaking the relatively weak hydrogen bonds between bases of opposing strands, whereas high pH deprotonates some of the bases, leaving fewer places where hydrogen bonds can form. DNA will renature if the conditions are reversed- for example, when temperatures or pH are lowered.

Know hybridisation can occur between complementary DNA and/or RNA molecules. 

When temperatures or pH are lowered, not only can two complementary DNA strands anneal to each other, but RNA can anneal to the DNA as well.

Be able to describe the difference between heterologous and homologous probes. 

Homologous probes are used to find identical genes, whereas heterologous probes are used to find similar genes.

Be able to describe the process of random priming.

Random priming is a technique used to label DNA molecules. The DNA is placed in a solution containing nucleotides labelled in some way- maybe they are radioactive or they have a chemical tracer attached to them. The DNA is then denatured and short primers are added on to the strand. DNA polymerase then creates a complementary DNA strand, using the labelled nucleotides to do so.

Be able to describe the Southern, Northern, and in situ hybridisation techniques and give examples of the uses of these techniques.

Southern blotting is a technique used to find out if identical or related genes are present within chromosomes. Firstly, the DNA to be studied is cut with restriction enzymes and run through agarose gel electrophoresis, a process described in my previous post. This is then blotted onto nitrocellulose paper. The DNA doesn't show up visibly on the nitrocellulose paper, so the paper is placed into a plastic bag along with labelled probes- sequences complementary to the sequence that is being searched for. The colour change or radioactivity produced by the labelled probe then aids in the identification of related sequences.

Northern blotting is a similar technique, but it is used to find out if genes are being expressed. To determine if a gene is actually expressed, Southern blotting is performed, but mRNA is used instead of DNA, as the presence of mRNA is usually a good indication that the gene is being expressed.

In situ hybridisation is a technique to determine where transcripts can be found within tissues or cells. A coloured probe binds to mRNA within the cells. This technique can be used to examine gene expression throughout development.

There is another hybridisation technique called fluorescent in situ hybridisation. In contrast to regular in situ hybridisation, fluorescent in situ hybridisation requires breaking open the cells and nuclei so that the chromosomes can be spread out. These chromosomes can then be denatured and fluorescent probes can be used to determine the location of particular genes on chromosomes.

Introduction to Cloning

While I was doing some practice questions, I realised that I was quite shaky on the details of cloning and hybridisation methods, so I'm skipping ahead to this section of the course.

Define a recombinant DNA molecule.

A recombinant DNA molecule is simply a molecule of DNA made up of different fragments that would normally not be found together in nature.

Know the properties of restriction enzymes.

Restriction enzymes, if I remember correctly, originate from bacteria. They cut DNA at specific locations, a function that is used in nature to protect bacteria from bacteriophages (viruses that attack bacteria). To prevent restriction enzymes from cutting the bacteria's own DNA, restriction enzymes work closely with methylases, enzymes that add methyl groups to certain bases in DNA so that the cell can recognise the DNA as its own.

Restriction enzymes only cut the DNA at specific sites. These sites are 4-8 base pairs long and are palindromic- that is, the sequence on the top strand is the same as that on the bottom strand, but reversed (since the strands run antiparallel to each other). When the strands are cut, 3'-OH and 5'-phosphate ends are formed. These ends can be "blunt" ends if, after cutting, the two strands on one side of the break are of even length and the two strands of the other break are also of even length. If this is not the case, then the ends are said to be "sticky" or "cohesive." They are said to have 5' overhangs if the 5' end juts out more, or 3' overhangs if the 3' end juts out more.

Know restriction enzyme-digested DNA with complementary ends can anneal.

After a strand has been cut to leave "sticky" ends, it can anneal to other cut strands that have a complementary sequence on their "sticky" ends. For example, if a strand is cut with a 5' overhang reading AATT, it can bind to an exposed TTAA on another cut strand. DNA ligase then fixes up the backbone.

Know how DNA ligase works.

DNA ligase is the enzyme that "patches up" any gaps in the backbone by covalently linking 3'-OH and 5' phosphate groups. They require ATP to carry out this process.

Know the properties of a plasmid vector and the three properties that make them useful for molecular cloning.

Plasmids are extrachromosomal DNA found in bacteria- that is, plasmids are DNA that aren't considered to be part of the bacteria's chromosomes. They are small, double-stranded and circular. Plasmids contain a multiple cloning site, or polylinker, which has several places where different restriction enzymes can cut. We can use this to insert other DNA into a plasmid for the purposes of cloning, which I will explain in a bit.

Know the steps in inserting a DNA fragment into a plasmid and amplifying it in bacteria.

To insert a DNA fragment in a plasmid, firstly you need to obtain a DNA fragment. You can do this by cutting DNA with a restriction enzyme. You can then cut the plasmid in the polylinker site using the same restriction enzyme. The fragment can then anneal to the plasmid DNA and DNA ligase can then stitch up the backbone.

Cloning plasmids requires a host organism, such as a bacteria. Plasmids can be inserted into bacteria by using heat shock or electroporation (exposure to an electrical field) to make bacterial cells more permeable to DNA. Once this has occurred, the bacteria can divide, producing many copies of the plasmid at the same time.

Be able to describe agarose gel electrophoresis.

Agarose gel electrophoresis is a technique that can be used to determine the length of the fragments generated from cutting the DNA. The length of the fragments can be added up to determine an approximate length of the entire genome.

In agarose gel electrophoresis, the DNA solution is mixed with ethidium bromide, which binds to DNA and RNA and appears fluorescent when viewed under UV light. This solution is then placed in the agarose gel solution. A current is applied to the solution. As DNA is negatively charged, due to its phosphate groups, the DNA migrates through the gel towards the positive electrode. Smaller fragments move more quickly than larger fragments, so at the end of a certain time period the location of the fragments (as seen under UV light) can be compared to fragments of a known length in order to determine the length of the DNA that you are interested in.

Know the characteristics of an expression vector that make it useful for the large scale production of proteins.

This is something that went clean out of my brain, so I revised it just then. Expression vectors are used for large-scale production of proteins. They include a highly active promoter region and a special tag that will be later used for isolating the protein in question. A commonly used tag is a series of bases coding for a cluster of histidine residues (a His tag).

To purify these proteins, affinity chromatography is used. In this technique, extract from the host cell is passed through a column that is filled with metal beads. His tags are more likely to bind to the metal beads, possibly due to the imidazole rings (I'll need to check this- I actually have no idea). As the extract passes through, eventually all that is left are the beads with the His-tagged proteins bound to them. The proteins can then be eluted (i.e. extracted) from the column by reducing the pH of the solvent (again, I'll have to find out why this is so).

Characteristics that would make an expression vector useful would probably be their ability to bind to metal beads or some other adsorbent. Another characteristic would be that they must be quite unique and different to other amino acid sequences commonly found in the cell so as to avoid contamination.

Cell Biology- Introduction and Energy Production

I'd better get on to studying for SCIE1106 (Molecular Biology of the Cell), because that unit is quite tricky. I'm not going to finish going through all of the CHEM1004 stuff, since the rest is either basic stuff that you should know before (like the structure of an atom and bonding) or stuff that requires diagrams to explain properly.

So now I'm onto the beginning lectures of SCIE1106. I can't believe that we only had four lectures with that guy- he covered so much content that it felt more like 6 lectures. Anyhow...

The first two lectures went from telling us what an atom is to explaining the basic properties of proteins, carbohydrates and lipids. (Yup, I told you that these lectures probably covered way too much...) These are topics that I've covered before, so let's take a look at the content of the next two lectures.

First up: similarities and differences between eukaryotic and prokaryotic cells!

Eukaryotic and prokaryotic cells are similar in that they all use more or less the same molecules, carry out the same basic chemistry and carry information in their DNA. However, they have several important differences.

The main distinguishing difference between eukaryotic cells and prokaryotic cells is that prokaryotic cells do not have a nucleus, whereas eukaryotic cells do (well, mature red blood cells don't, but they're pretty much the only exception). Eukaryotic cells store their linear DNA in the nucleus, whereas prokaryotic circular DNA just floats around in the cytosol. In fact, aside from not having a nucleus, prokaryotic cells generally do not have any organelles at all, whereas eukaryotic cells have several membrane-bound organelles which carry out various functions in the cell. Another difference is that while both cells have ribosomes, eukaryotic cells have larger ribosomes than prokaryotic cells.

Cells can derive energy from different sources. Organotrophic cells derive energy from organic molecules, phototrophic cells derive energy from light whereas lithotrophic cells derive energy from inorganic molecules.

Organelles

As mentioned above, eukaryotic cells have many different organelles- different membrane-enclosed structures where various processes take place. I've already written a bit about membranes in an earlier post- I might write another one later more specific to the content of this unit.

Nucleus

The nucleus is the place where most of the eukaryotic DNA is stored. Some of the DNA is present as heterochromatin, which is highly condensed throughout the entire cell cycle, while the rest is present as euchromatin, which is not condensed until mitosis. The nucleus has a double membrane which is continuous with the membrane of the endoplasmic reticulum. The membrane has several pores through which substances can pass through. Within the nucleus, there is a structure called the nucleolus, in which ribosomes are produced.

Ribosomes

Ribosomes are packages of protein and ribosomal RNA (rRNA) found in the cytosol and endoplasmic reticulum. They are the site of protein synthesis and are made up of two subunits: a small and a large subunit. Eukaryotic ribosomes are larger than prokaryotic ribosomes- eukaryotic ribosomes are 80S while prokaryotic ribosomes are 70S. (Sorry, I can't remember exactly what the "S" was meant to stand for right now, aside from that it's a form of measurement. Something to do with sedimentation or something like that.) Ribosomes located within the mitochondria and chloroplasts are also 70S (it has been hypothesised that mitochondria and chloroplasts were originally bacteria that were engulfed by eukaryotic cells).

Mitochondria

Mitochondria are often referred to as "the powerhouse of the cell," as they produce ATP (adenosine triphosphate), a form of energy that the cell can use. (One common misconception is that mitochondria produce energy. They do not produce energy, as that would be breaking the law of conservation of energy. Mitochondria simply convert energy to a form that can be used by the cell.) Mitochondria, like the nucleus, have two membranes: a smooth outer membrane that is permeable to small ions and molecules, and a folded inner membrane which is impermeable and has transport proteins to carry solutes across. The folds of the inner membrane are also known as "cristae," and they are where ATP synthesis takes place.

Aside from having their own 70S ribosomes, mitochondria also have their own DNA. They also have their own RNA as well. This DNA codes for enzymes required for the reactions that produce energy.

Energy production is quite a complex process. It starts in the cytosol, where carbohydrates and some amino acids are oxidised to form pyruvate. This process, known as glycolysis, generates some energy molecules. Pyruvate can then enter the mitochondria, along with fatty acids. Both pyruvate and fatty acids are then converted to acetyl CoA (which is essentially an acetyl group attached to coenzyme A).

In the mitochondria, acetyl CoA is repeatedly oxidised in a cycle known as the citric acid cycle. (Some amino acids can also enter this cycle directly.) CO2 is produced in this cycle, and the mobile electron carriers NAD+ and FADH are reduced to NADH and FADH2, respectively. (I mentioned these electron carriers in an earlier post for CHEM1004.)

NADH and FADH2 carry electrons to the electron transport chain (ETC), located in the inner membrane of the mitochondria. Electrons from NADH first enters Complex I (NADH dehydrogenase complex) of the ETC, are carried by ubiquinone (Q) to Complex III (Cytochrome b-c1 complex), are carried by cytochrome c (c) to Complex IV (cytochrome oxidase complex), where they finally reduce oxygen to form water. (This is why oxygen is required for aerobic respiration in the mitochondria- if insufficient oxygen is present, the cell makes do with energy produced from glycolysis.) The energy released when these high-energy electrons travel through the ETC transports protons into the space between the two mitochondrial membranes. This generates a proton gradient as there are more protons in the inner membrane space than in the mitochondrial matrix. The protons move through ATP synthase to get back to the matrix. As they move through ATP synthase, ATP is produced.

Electrons from FADH2 travel through the ETC in the same way, but instead of entering Complex I, they enter Complex II (succinate dehydrogenase). Ubiquinone then takes these electrons to Complex III.

Chloroplasts

Chloroplasts are organelles only found in plant cells. Like mitochondria, they are also likely to have originated from prokaryotic organisms that were taken up into eukaryotic cells. They also have their own DNA, RNA and 70S ribosomes.

Chloroplasts are sites of photosynthesis, or converting light energy into a form that can be used by the cell. They do this by harvesting light and producing carbohydrates.

Chloroplasts, like mitochondria and the nucleus, have double membranes. The two membranes have similar properties to that of the mitochondria: the outer membrane is smooth and permeable to small molecules whereas the inner membrane is impermeable and has transporters to transport solutes across it.

The inside of chloroplasts contains stacks of thylakoids, which are formed by the folded internal membrane. These stacks are also known as grana, and they are where light is harvested. The lumen is the space between thylakoids in a granum. The stroma is essentially the rest of the inside of the chloroplast.

Now it's time to look at how chloroplasts harvest light! This should be a challenge seeing as I didn't really learn it that well the first time. At least now that I understand ATP synthesis, it shouldn't be too hard, right? Right?? Well, we shall see...

The thylakoid membranes contain special pigments that can collect light. This light energy is converted to NADPH and ATP, though unfortunately the slides don't go into too much detail into how they do this. I did find a good website though- http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/L/LightReactions.html.

The understanding I have is that the electron transport chain in chloroplasts has two "photosystems" called I and II. These have antenna pigments that can collect light (as I mentioned above). When Photosystem II absorbs a photon of light, it removes an electron from a molecule called P680. located inside Photosystem II. After an electron has been removed, P680 is electronegative enough to pick up electrons from water in the lumen. These electrons are picked up by plastoquinone (PQ) to the cytochrome b6/f complex. Electrons provide this complex with energy to transport protons from the stroma to the lumen, creating a proton gradient. Electrons at the cytochrome b6/f complex are then picked up by plastocyanin (PC) to Photosystem I. The electrons then pass through ferredoxin (Fd) before ultimately reducing NADP+ to NADPH. Additionally, the protons from the proton gradient that was formed earlier power ATP synthase as they move back into the stroma- just like ATP synthase in the mitochondria.

Sorry if that was overly confusing :(

After NADPH and ATP are produced, they can power the synthesis of carbohydrates from atmospheric CO2. This process, known as the Calvin cycle, takes place in the stroma of chloroplasts. Rubisco, which is apparently the most abundant enzyme in the world, catalyses the first step of this process. Fortunately, we don't need to go into much detail about this for now.

Endoplasmic Reticulum

The endoplasmic reticulum is a network of sacs and tubules that extends throughout the cytosol. It can be divided into two sections: the rough endoplasmic reticulum, which contains ribosomes for protein synthesis, and the smooth endoplasmic reticulum, where lipids are produced. The cisternae (tubules) of the rough endoplasmic reticulum are parallel and flat, whereas the cisternae of the smooth endoplasmic reticulum are tubular and branching in comparison.

Golgi Apparatus

The Golgi apparatus is a specialised section of the endoplasmic reticulum in which proteins are modified before being packaged and sent to different locations. The cis face of the Golgi apparatus is adjacent to the rest of the endoplasmic reticulum, whereas the trans face faces towards the cell membrane. The most common modification that takes place within the Golgi apparatus is the addition of carbohydrates to form glycoproteins (i.e. proteins with carbohydrate chains added).

Vacuoles

Vacuoles are seen mainly in plant cells, though if I remember correctly animal cells may have some smaller vacuoles as well. They have many functions, including degradation, detoxification and storage.

Peroxisomes

Peroxisomes are small organelles containing oxidation enzymes. They perform detoxification functions in both animal and plant cells. In plants, peroxisomes are also sites of photorespiration (carbon recycling) and conversion of stored fats into sucrose.

Cytoskeleton

The cytoskeleton is a topic that I have already covered in an earlier post, though again I may write another post more relevant to this unit.

Phew! I'm going to go rollerblading now!