Look the equations on this Blog for Entropy will be AMAZING! :D

Consider $\sigma : \mathbb{R}\to\mathbb{R}$ a linear transformation. $$ \int_0^\infty e^{-x}dx = 1 $$ S=dh/dT $S=\frac{dH}{dT}$ $\sum_{i=1}^\infty \frac{1}{e^i}$

Enhancer Odds and Sods

Transfection Assay: Activity of Enhancers

Comparison of a virus with a specific gene and an enhancer, and a virus with a specific gene and no enhancer can reveal the activity of the enhancer by seeing qualitatively how much more of the mRNA is transcribed by the trial with the enhancer present.

Eukaryotic Genes Are Regulated By Many Transcriptional Control Elements

Pretty much the what the heading of this sentence says. Some of elements include the TATA box, the promoter proximal elements, enhancers - and remember enhancers can be anywhere!

Something About Yeast

A common regulatory element that acts like an enhancer in yeast is called UAS. The TATA box in yeast is 90 base pairs from start site. Your life is now complete from knowing these details. :)

Deletion Analysis: Mapping Enhancers

Deletion Analysis works to find enhancers that can be many kilobases away from the start site of a gene. By taking a longer stretch of DNA sequence, and using a restriction enzyme to cut at various sites of the sequence, when the modified sequence inserted into a plasmid vector and placed within the cell, the amount of reporter gene indicates whether transcription has been affected. Comparison between transcription of various modified lengths of the sequence from longest sequence to shortest sequence reveals the location of the enhancer in an interval of bases. This technique is not as precise, but it still finds the region of the enhancer element, much in the same manner that a linker mutation technique does, by seeing where transcription stops. The region of the enhancer element is found within the difference of the bases of the closest longer sequence trial and the sequence where the transcription stops.

Linker Scanning Mutations

To locate promoter proximal elements in a short stretch of DNA up to around 100 base pair upstream of the TATA box, an experiment involving linker-scanning mutations can be used. A region of DNA that sequences a reporter gene is cloned several times. The clones are altered by addition of scrambled nucleotide sequences known as linker scanning mutations that are introduced from one end of the region of the sequence to the other. This is only done in a short stretch of the DNA to pinpoint the exact region of the promoter proximal elements. Then when the sequence is placed back into the cell via a plasmid vector, the rate of transcription (how much mRNA is produced) is measured by the amount of proliferation of the reporter gene. If transcription occurs, the region does not contain a promoter proximal element. If transcription has stopped or is reduced, the region of the linker mutation is that of a promoter proximal element, and the bases can then be identified.

Eukaryotic Gene Expression: The TATA Box and Other Promoters and Enhancers

Meet the TATA Box

First thing is that structure-wise this structure doesn't have too many silly nomenclature tricks up its sleeve. For once, the name is what it says it is, a TA rich region on a DNA sequence. Functionally, it positions the RNA polymerase on the DNA sequence for transcription, acting similarly to an E coli promoter. The TATA box is located around -35 to -25 base pairs upstream of the start site. So if some clever multiple choice question asks "Is the start site at the TATA box?" the answer is "No, it is downstream." "Do all genes have a TATA Box?" Again, "No, only those that have high rates of transcription in the cell." We will see plenty of other wonderful ways genes can be transcribed without a TATA box, albeit not in much detail. They will be discussed shortly.

How TATA is your TATA Box? 

The TATA box is called a consensus sequence, and it is highly conserved among various genes in various organisms. However, each of the bases (A, T, and also G, and C) have a certain frequency of being in the ideal location "TATATATA" of the TATA box.

The third base has a 100% frequency of having a T, but the other bases are not as clear cut, with the first base being 83% likely of having a T, the second being 91% likely of having an A, and the others having 100%, 95%, 33%, 97%, 36% and 41% for the ideal base be it T or A respectively. There is a 40% probability of having a G in the last position.

This looks like memorization hell, and quite frankly it is, but the take-home message (which never shows up in multiple choice for this course for the record, oops that might have been sass) says the following: the TATA box is highly conserved, however different bases may be present in its sequence. I take back what I first said about the nomenclature, a better name would be the "most likely but not quite always TATA box."

Alternatives to the TATA Box

Initiator Element
Not much is known or considered important to relay at the undergraduate level about initiator elements except that a C is found in the -1 position and an A is found in the +1 position. I am really not sure how they did experimental testing to figure out these details, except it really must occur with a significant (or relatively significant) degree of frequency in the sequence, so if anyone knows and the explanation isn't too complicated that would be cool.

CpG Islands
These are CG rich areas of 20-50 base pairs within 100 base pairs of the start site region of a gene. These genes often have multiple start sites for transcription in a 20-200 bp region, and have neither a TATA box, nor initiator elements.

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Promoter Proximal Elements

These are sequences within 100-200 base pairs of the start sequence that aren't the TATA box or any of the above sequences mentioned. They can be cell-type specific (not universally conserved).

Enhancers 

Enhancers can be quite far away from the gene they enhance - even greater than 50 kilobases away! Their location may be upstream from the promoter, downstream from the promoter, within an intron, or downstream of the final exon of the gene. As one of my favourite Beatles songs likes to say, they can be quite literally be "here, there, and everywhere." The direction of the enhancer doesn't matter! Also, like promoter Proximal Elements, they are often cell type specific.

Difference between Promoter Proximal Elements and Enhancers? 

Recall that it is with a human categorical bias that we organize these components of the cell. So, the distinction between promoter proximal elements and enhancers is not clear cut. For the purposes of the course I am taking, if something is within 100-200 base pair of the start sequence and helps initiate transcription, we'd probably call it a promoter proximal element - but who knows it could just as easily be an enhancer, or perhaps it is both!

**The next post will continue with an explanation about finding Promoter Proximal Elements with linker scanning mutations, and deletion analysis, and then there will also be another post about Enhancers and their effects on transcription.

The Epic Tale of the Man who Stepped on Glass

This post took awhile to write and is hopefully accurate. After sifting through lecture notes, my textbook, and internet articles, "The Epic Tale of the Man who Stepped on Glass" compiles the entire immune response as required for understanding of someone enrolled in a first-ever physiology class. While reading, I found that often lists of cells would be provided and then lists of processes. This "tale" takes on both concurrently! Please do note that the focus is on bacterial infection, and not viral infection. However, there is a note at the end, which explains a bit about viral infection as well. 

Once upon a time, there was a man. He may have been a good man or a bad man, I'm not entirely sure, however, the qualifier in this case is irrevelant. On one fateful day the man accidentally (or consciously, but it is highly unlikely this was the case) stepped on a piece of broken glass. The glass had shattered onto the ground, and as he was barefoot, and one of the fragments pierced his skin. http://www.youtube.com/watch?v=Rw0eIG_9tLY

Apart from the man's howl of anguish as the blood dripped down the arch of his foot, other courses of action were taking place in the man's body. Yes, his foot was throbbing and swollen, but at the cellular level, things were at work. The glass had broken through the first line of defence, the coverings of the body. The skin and the mucous membranes usually provide an unpleasant environment for the living conditions of microorganisms, but the glass had successfully penetrated the skin. Little did the man know that the glass had been infected by a bacteria, so the next line of defence had to be called into action.

The fixed tissue macrophage, present at the site of the infection, is the first cell to be involved in the innate immune response. They have toll-like receptors (TLRs), which are attracted by the PAMPs produced by the bacteria. PAMPs, pathogen-associated molecular patterns, are lipopolysaccrides or peptidylglycan molecules present on the surface of bacteria, which are absent in mammals, and are therefore considered foreign. The fixed tissue macrophage will take up some bacteria by phagocytosis, and emit a signal called the MDNCF, the monocyte derived neutrophil cytotaxtic factor, which recruits neutrophils to the site of the infection.

The neutrophils reside in the blood, and travel to the capillaries, but in order to pass through the capillary wall, some modifications are necessary. The neutrophil has to change its receptors and the endophilial cells of the capillaries need to change their receptors. After this, the neutrophil can then adhere to the endophilial cell surface, where it rolls along the capillary cells until it gets to a point where it can squish in between cells called diapedesis. From there the neutrophil travels to the infectious site.

When the neutrophil encounters a bug, it takes it in by phagocytosis. The microbe contacts the membrane of the neutrophil, which envaginates to form a pocket, which then pinches off separately from the membrane to form a phagosome. This is analogous to the foreign particles of food entering your body in a separate pouch known as the stomach, so that digestion does not interfere with the internal processes of the body, or in the case of the neutrophil, so digestion does not interfere with the processes of the cell. A lysosome, filled with digestive enzymes fuses with the phagosome to make (you guessed it!) a phagolysosome, which breaks up the macromolecules into fragments, and oxidative death also occurs. The neutrophil dies in the process of killing bacteria, and then new neutrophils come along and ingest bugs and also die. In the process of dying, the neutrophil extrudes DNA, chromatin and protases, which form a neutrophil extracellular trap or NET. This keeps the bacteria from spreading and contains the bacteria. The dead neutrophil and bugs under skin make pus.

PAMPs can activate complement, which can in turn lead to the killing of pathogens in what is referred to as the alternative pathway. This can be done by a Natural Killer cell, which is also important in the innate immune response. They have a default response of "kill" when they encounter a cell. This function is so strong, they will even act against the body's best interests, and create an auto-immune response against "self." If the ligand of the Natural Killer cell is activated, unless it is turned off by the MHC class I molecule which is present on all nucleated cells, it will kill that cell, which is a problem of auto-immune disease. In many cancerous cells, the MHC class I molecule is faulty, as well as viruses can change the MHC class I molecule. Normally, it is turned off.

The next cell in after the neutrophil is the dendritic cell, which has two functions: phagocytosis and synthesis. It travels with the bacteria on its dendrites to the nearest lymph node, which in the case of the site of infection of the man's foot, happens to be in the groin. The dendritic cell links the innate immune response to the adaptive immune response, the third line of defence. The adaptive immune response is acquired and specific will the humoural aspect of the immunoglobulin mediators. It is also cell-mediated by T cells or lymphocyte effectors.

The adaptive immune response is specific to the antigen and epitope (one site of the antigen where the antibody attaches itself). The antigen presenting cells (APCs) link the antigen by means of a MHC (major histocompatibility complex) class molecule and a peptide. They may be dendritic cells, a macrophages or a B lymphocytes. The MHC molecule is unique to a specific person. There are two kinds of MHC molecules, class I and class II. Recall that class I is present on all cells in the body to turn off the Natural Killer cells. However, class II is only present on three molecules: the dendritic cells, the macrophages, and the B lymphocyte, because it is the antigen presenting molecule. After the antigen is broken down into peptides, it is presented on the dendritic cell, on the MHC class II molecule. The mjaor histocompatibility complex is called the HLA system, for Human Leukocyte Antigens, because that is where it was first seen.

Another way of phagocytosis is done by the dendritic cell. The dendritic cell goes through the same procedures as the neutrophil, except in the phagolysosome, there is the synthesis of the MHC class II molecule with an antigen fragment. The complex then comes to the surface of the cell. This holds any antigenic peptide, without specificity.

Now, before we move into the next steps of the adaptive immune response, it is necessary to take a trip back into the man's past, or more particularly, the past of his T- and B-cells. The T-cells and B-cells are made first in the yolk sac of the fetus, then in the liver and spleen after the first trimester, and finally in the bone marrow after birth. Some of these cells travel to the thymus to become T-cells. In the environment in the thymus and bone marrow, the cells mature, differentiate, and gain specificity for many possibilities of antigens.

Out of all the T-cells which mature in the thymus, only 5% leave the thymus to the blood, where as the rest of the 95% of the cells die. The vast majority of the cells produced in the thymus are anti-self, and must be eliminated to prevent a future auto-immune response. The 5% survivors have specific reactivity to only one peptide.

In the lymph nodes, the B-cells live in the portal part of the lymph node, and the T-cells are located in the madorary part of the molecule. When the dendritic cell leaves the site of infection, it travels to the site in between the B-cells and T-cells. Since the T-cell has a specific receptor that was specified in thymus pre-birth, it will see only one peptide material, one amino acid. This is the MHC peptide, which is recognized by the T-cell receptor, and thus has specificity. The CD4 molecule is involved in binding T-cells to the MHC II region of the MHC-peptide complex on the APC (antigen presenting cell).  As a double-checking mechanism, the T cell synthesizes CD28, which is a co-receptor. If CD28 binds to the B7 molecule on the dendritic cell, the antibody will be produced. However, if the co-receptor is not bound, then no antibodies are made, and the organism becomes tolerant to the protein. Note that the CD28 - B7 binding is not specific.

Also, on the surface of the T-cell is a receptor called CD40L (the L stands for ligand), which is associated with an enzyme that is a ligase called CD40. The B-cell binds to the CD40L, and then the T cell produces at least three interlycukins called IL 4, 5,  and 6, which cause proliferation, further maturation, and induction of the antibody. Next occurs a puzzling step where the B-cell's surface immunoglobin with antibody specificity binds to the bug on the dendrite. This is odd as the B-cell sees the whole bug, but the T-cell sees only a specific protein, yet together they make one antibody. Does this happen at the same time or in sequence? This is a mystery.

Now, both the T-cell and the B-cell have identified the bug as an immunogen, so what happens next? First, it is necessary to consider the structure of the immunogobulin antibody molecule. The molecular was initially found through electrophoresis, where the albumin molecule (a protein in blood), was seen to be homologous, but the other gobulin molecules where found to be heterogenous. The antibody activity is mostly found in the gobulin molecules, with the majority of the activity in gamma globulin and some in other classes. The immunogolbulin molecule has a tetrapeptide structure with four chains, two which are identical and heavy, and two which are identical and light. The light chains give the immunogolbulin its type, whether it is lambda, or kappa. The heavy chains determine the class of molecule: delta, my, gamma, alpha, or epsilon. Interchain and intrachain disulphide bonds are present between and within all chains.

The Fab of the immunogobulin is the antigen binding fragment, which is found at the hypervariable region, and it is divalent, so two antigens can bind. The Fc region is a crystallizable fragment which determines the biological activity of the Ig class. For example, the IgM exibits complement binding (explanation of complement forthcoming!) and is a pentameric molecule. The IgM is the first immunogobulin to respond, but it is inefficent and undergoes class switching. The IgG is present for placental transfer until the fetus can make its own immunoglobulins, is also involved with complement binding and is a dimeric molecule. Also, the IgA is involved with secretory properties including the MALT, and ensures that foreign substances that are encountered from the nasal cavity to the lungs, the digestive tract, do not cause the body to freak out. The IgE deals with mastocytophilic properties, or allergies.

After all of that information about immunoglobulins, it is important to recognize that they are often released by plasma cells, which does not sythesize immunoglobulin (antibody), but just secrete the immunoglobulins (antibodies) made during the earlier part of its B-cell life. The pro-B cell does not have surface immunoglobulin, but the pre-B cell does have this surface immunogobulin that is produced in the bone marrow, and is specific and waiting for the particular antigen to arrive in the cell. The plasma cell loses the surface immunogobulin and secretes antibody from the cell itself.

Four genes determine the structure of the heavy chain message, and each gene has multiple alleles. This is an interesting concept where randomness can actually lead to specificity. There are three regions to choose alleles from on the heavy chain: the variable region, the diversity region, and the constant region, plus an alpha, delta, mu, gamma, epsilon, determining the class. The arrangement of the immunogobulin is carried out by enzymes called the rearrangement activation genes or RAG, and the process happens in the thymus or bone marrow. This allows for hundreds of millions of different specificities, many of which may remain dormant and never be used, and others (95% of all T-cells) which may have a tendency for auto-immune response and be anti-self, so thus are knocked off by the thymus.

So, the helper T-cell has recognized the peptide linkage and the B-cell has recognized the bug, now what? Antibodies are released and then the Fab binds to the antigen, and can start the classical complement pathway (if bound to an IgM or IgG). This is a release of many protein C factors, in a complement cascade. If the factors proceed all the way to factor C9, a hole in the bacterial membrane forms, and the bug explodes. However, if the factors proceed only to C3 and C5, a so-called alternative pathway occurs, which turns on the Innate Immune Reponse, and enhances phagocytosis. Neutrophils are called in to phagocytose the bug, and then the neutrophils die releasing the net. Macrophages then arrive to clean up the pus.

In conclusion, after the immune response has finished, memory cells (either B-cells or T-cells involved in the response) are stored in the apical light zone of the lymph node. The rest of the B-cells commit suicide, called apoptosis.

Note that in the response described above, the T-cell involved was a TH2 cell, or a T Helper 2 cell. Had this not been a bacteria infection, but that of a virus, the TH1 cell would have interacted with a dendritic cell that had taken up a pathogen by means of an MHC II bound to an epitope and a TCR (T-cell receptor) of CD4+. They also have co-receptors of CD28 which binds to B7 on the dendritic cell, as a double-checking mechanism. This is exactly like the TH2 cell. But here is where the process differs: the TH1 cell releases IL-2, TNF, and INF which stimulate the Cytotoxic T-cell. The Cytotoxic T-cell's TCR which is specific for the antigen binds to the virus by recognizing an MHC class I molecule linked to the virus. This only occurs, however, if the MHC class I molecule is linked to CD8. Hence, cT cells are often described as being CD8 positive.

Prokaryotic Gene Expression

Transcriptional Control

The major mechanism for controlling protein production in the cell is determined by which genes are transcribed to encode a particular protein. This is important as the structure and function of the cell is determined by the proteins it contains. The cell regulates the proteins and speed of which they are produced by repressing and activating the gene in question.

repressing a gene: the corresponding mRNA is transcribed at a low rate (meaning little to nothing in the cell)

activating a gene: the corresponding mRNA is transcribed at a much higher rate (meaning up to 1000x or more RNA is transcribed)

Purpose of Transcriptional Control...

...in Single-Celled Organisms

Transcription of genes is regulated to adjust to changes in the nutritional and physical environment. The cell will produce only the proteins required for survival and will proliferate under the particular environmental conditions it experiences.

... in Multicellular Organisms

Transcription of genes is regulated to ensure coordination during embryonic development and tissue differentiation. Again, the level of organization is more complex.

Operons are Efficient for Control

Recall that operons are sequences that encode enzymes in a row that are involved in a particular metabolic pathway or proteins that interact to form a large multi-unit protein. This is characteristic of procaryotes, as eukaryotes have their proteins in different regions of the chromosome separated by large uncoded regions called introns. Point being, in E coli, half of the genes are clustered into such operons, with the trp operon coding for five enzymes needed in the biosynthesis of tryptophan, and the lac operon coding for three enzymes needed in the biosynthesis of lactose.

All genes on an operon are coordinately regulated, meaning that they are repressed and activated to the same extent, which is efficient and economic for controlling transcription regulation in bacteria. The regulation happens through RNA polymerase and specific repressor and activator proteins. To initate transcription, RNA polymerase must associate with a sigma factor, most commonly . These sigma factors are not conserved in eukaryotes.

Function of Sigma Factors

Sigma Factors recognize specific DNA sequences as promoters and recruit RNA polymerase which is a lot simpler sounding than that whole eukaryotic preinitiation complex razzmatazz. After transcription is initiated the sigma factor peaces out, or is released from the promoter upstream of the start site.  recognizes the sequence TTGACA (in the -30 region) ...15-17 bps... TATAAT (in the -10 region). On the other hand, recognizes a very difference sequence as it is involved with metabolizing nitrogen. Eg: the regions on the DNA which these sigma factors recognize are diverse.

The E coli Lac Operon Saga

The E coli lac operon exists in a repressed state.

The E coli Lac Operon encodes the three enzymes required to metabolize lactose in the cell. However, the cell much prefers to get its energy from glucose, so the enzymes will only be transcribed if there is a) high lactose and b) low glucose in the cell. Normally, this is not the case, so the lac repressor binds to the operator, blocking the start site, and the promoter is ready for polymerase to bind, no transcription occurs.

De-repressing the lac operon. 

However, when lactose enters the cell in high qualities, it binds to the lac repressor, causing a conformational change and for it to release from the operon. Then binds and recruits the polymerase to the promoter. Transcription occurs, until lactose levels are low once more in the cell, and then the lac repressor will again bind to the lac operator. It should be mentioned though, that transcription levels of the proteins are low for this scenario as glucose (the preferred metabolite) is still present in the cell.

Activating the lac operon. 

If both glucose levels are low in the cell, and lactose levels are high, then something else happens with the operon. cAMP is produced by the low glucose levels in the cell and then binds to CAP (catabolite activator protein) from there, the complex binds to a site upstream of the promoter called the CAP site. The lactose binds to the lac repressor, causing the conformational change and release from the operator, then the polymerase binds to the promoter complexed with . The cAMP and CAP complex greatly stimulate the rate of transcription.