Review of DNA dynamics

Week 2 assignment

General rules that must be followed:-

1- Remember to number the answers to each question.

2- Be kind to my eyeballs. You do NOT have to write pages and pages and pages.
Be CONCISE. However, there is sometimes a fine line between being concise, and
being incomplete. So, go directly to the point of the question – go DIRECTLY. Your
answers will always be evaluated on quality, rather than quantity, of information.

3- Please do not include the questions themselves in your submission. Either delete the
questions, or create a separate document.

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Topic 1 – A review of DNA dynamics.

1. The following are excerpts from the 1953 Watson and Crick paper:

a. “It has been found experimentally that the ratio of the amounts of adenine to thymine, and the ratio of guanine to cytosine, are always very close to unity for deoxyribose nucleic acid”.
b. “It has not escaped out notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”.

Were they really ahead of their time? Tell whether each statement is still completely true today, or, if it is not, tell why it needs to be corrected.

2. Look at the different isoforms of DNA. Assume each of these is capable of synthesizing a primary transcript (pre-mRNA). Would the splicing out of introns be easier (or harder) depending on the DNA isoform source? Or would splicing be the same no matter where the pre-mRNA came from? Support your choice with good science.

3. “Initiation”, “elongation”, and “termination” work in both replication and transcription. Which of the three has virtually the same action in both processes? There is no right or wrong answer, but support your choice with good science.
Topic 2 – The incredible GRANDE summary of peptide synthesis.

4. In the Biologynews.net article, several functions are suggested for the CNS’s, those segments of DNA that do not code for protein. One of these is that they interact directly with RNA. Is this interaction beneficial, or is it a detriment? Choose either stance, but support your logic.

Topic 3 – Something special about RNA. Please choose either 5a or 5b. Do not answer both)

5a. RNAi therapy has worked well with palliating macular degeneration, even though many genes cause the condition, many of which are not that well-known. On the other hand, this therapy has been suggested for Parkinson’s Disease, where the causative gene is well known, and easily targeted. Do you think this therapy would also work JUST AS well for Parkinson’s disease? Why, or why not? Be sure you include the action of RISC in your response.

5b. RNAi therapy has been cited as a great way to cure infectious diseases. Do you agree with this? Why, or why not? Be sure you include the action of RISC in your response.

Topic 4 – You’ll be amazed at the definition of “the basics”.

6. In addition to simply amplifying DNA, PCR is also used to diagnose genetic disorders by revealing the presence of abnormal DNA sequences. Choose either “a” or “b” and support your choice with good science:

a. Tell why PCR is a better technique than a Southern blot to determine if a patient has an abnormal DNA sequence.
b. Tell why a Southern blot is a better technique than PCR to determine if a patient has an abnormal DNA sequence.

7. You have a choice of using cDNA to either CURE or DIAGNOSE a human genetic disorder. Would it be more useful as a cure, or for a diagnosis? Support your opinion with good science.

( Topic 1) A Review of DNA Dynamics
Let me warn you – this week’s set of materials contains a lot of chemistry. SO, if chemistry is not your strong suit, you will need to do this one bite at a time.
In April of 1953, a one-page article was published in Nature by James Watson and Francis Crick (Crick passed away about eleven years ago — but Watson is still kicking around. I had the pleasure of seeing him at a conference luncheon that I attended – he is 88 years old, eats like the world food supply is about to disappear, and makes the Mad Hatter seem sane. BUT, he becomes incredibly lucid when you talk science with him. He is now, and always has been, an incredibly brilliant scientist.
At the time, this publication was not highly regarded, kind of “ho-hum”. It is now a monumental landmark in all of science, as it was the first one to describe the structure of DNA accurately as a double helix, eventually resulting in a Nobel Prize for them. Just to see how far we have come, and how amazingly accurate they were with early 50’s technology, I want you to pause now and read the original paper by Watson and Crick, published 60 years ago. Click on the following link, and be prepared to see how far ahead of their time W&C were. Do you think they were correct when they surmised that DNA might be of “considerable biological interest”?
Watson and Crick’s landmark paper
(http://www.nature.com/nature/dna50/watsoncrick.pdf)

Up until June, 2000, when the first “rough draft” of the human genome project was released, this was the single most important discovery in molecular biology in the 20th century. The genome project absolutely puts the discovery of the double helix in second place (or third place if you include gummy bears). It is rather interesting that the human genome project was completed in April of 2003, exactly 50 years after the landmark publication in Nature.
Now for a word from the lawyers. Watson and Crick described the molecule based upon X-Ray diffraction work done a few years earlier, mostly by a scientist named Rosalind Franklin. In fact, if you look back at the paper, the references DO cite Ms. Franklin’s work. So, why didn’t she get any credit? Why isn’t it called the Franklin model? Back then, there were hardly any female molecular biologists. In fact, it was very hard for female scientists to earn any credibility, much less publish in recognized journals. So, she gratefully allowed Watson and Crick to use her findings, and —- well, the rest is history. Unfortunately, Ms. Franklin died of ovarian cancer at age 37. This happened four years before W&C received the Nobel Prize for their (?) discovery. Times HAVE changed!!! I have included a link below – this gives you the story behind one of science’s most brilliant people, who, like Gregor Mendel, never got the recognition that was highly deserved. Please keep an open mind as you read it. Click:
Rosalind Franklin – the REAL DNA pioneer?
(http://www.sdsc.edu/ScienceWomen/franklin.html)

Keep in mind that I am about as far from being a physicist as one can get. So if you know a lot of physics, try not to laugh, as I provide my amazingly scholarly interpretation of X-Ray diffraction. Here goes:
The procedure works by the diffraction (deflection???) of X-rays off a molecule, recording the bouncing pattern onto an X-ray plate. The shape of the molecule determines the pattern that the X-rays make on this X-ray plate. How’s that for sophisticated physics???
The following is the original image obtained by Rosalind Franklin, with a little help from a reasonably smart fellow named Linus Pauling. The autograph on the right indicates that this is a rendering from the two of them (“Franklin & Pauling”). The scribbling below their names says: “Sodium deoxyribonucleate, Type B”. So, it is actually the salt of the acid; as we will see, the salt is the usual form of existence for DNA.

This is, very obviously, a double helix (sure it is)!!! Actually, you can see some symmetry in the diffraction pattern. The relationship between this symmetrical pattern, and the symmetry in DNA, is a little beyond my area of expertise.
Now that you had a look at this symmetrical molecule from a rather unique perspective, here are a few more things about it. These facts reflect the latest information (I hope – I try to keep up, but it’s hard sometimes) about the molecular architecture of DNA. You might find yourself saying that “this is only important if you are actually studying the chemistry of the molecule and nothing else.” However, integrating these biochemical facts into material discussed later in the course will make it more relevant clinically. I promise.
1. Location of DNA in the chromosome.
Point of information: In your average, typical, run-of-the mill chromosome, did you know that there is actually more protein than nucleic acid? That’s right. The proteins and nucleic acids are organized into molecular packages called nucleosomes. A nucleosome is a series of eight globular proteins, or globins, called histones. The arrangement of these proteins is therefore called a histone octomer. The DNA loops around the histone octomer. Each looped sequence of DNA contains about 200 base pairs. The octomers are linked together by short lengths of DNA logically called linker DNA. And, if that is not messy enough, the linker DNA is attached to non-histone proteins, which form a background matrix called a scaffold.
Below is a sketch of a nucleosome. The colored globes are histone proteins. Because of the non-three-dimensional rendering, you can only see six of the eight, but there is one more blue and one more orange histone hidden from view:

The packaging of DNA into chromatin (review what chromatin looks like in interphase cell nuclei) allows the DNA of human cells (about 2 meters in length if stretched out) to fit into a nucleus with a diameter of only 10 µm. The basic repeat element of chromatin is this DNA-protein nucleosome, which consists of 200 base pairs (bp) of DNA wrapped 1.7 times around an octomer of histone proteins (two copies each of core histones H2A, H2B, H3, and H4). I am not a chemist – I am amazed how accurate science can be with this! Continuing, the core histones contain another, tri-helical, histone fold domain that mediates histone-histone and histone-DNA binding.
Isn’t that last paragraph a doozy? Yes, it is English. What it boils down to, is that proteins with enzyme-like properties allow histones to bind to each other, as well as to the associated DNA. Anyway, nucleosomes, connected by about 20 to 60 bp of linker DNA, form a 10-nanometer “beads-on-a-string” array, which can be compacted further into a “30-nanometer” chromatin fiber. Whereas the three-dimensional structure of the nucleosome is known in exquisite detail, the structure of the higher order 30-nm chromatin fiber is poorly understood.
Taking this one level higher, the nucleosomes are arranged in the chromosome as follows. Note that there are additional histones associated with the linker DNA:
So, to summarize the different levels, examine this diagram:

Interestingly enough, it was not until as recently as the early 1980’s (yeah, given the knowledge we have, we can call that “recent”) that the exact relationship between the DNA molecule and its location in the chromosome was actually confirmed.
2. Location of DNA in the mitochondrion.
It has been known for many decades that the mitochondria have their own, independently functioning DNA. A detailed discussion of mitochondrial DNA (mt-DNA) is coming up in a future week, so for now, we will just peek ahead a little. There are a multitude of structural and functional differences between “classic” nuclear DNA, and mt-DNA. It is known that mt-DNA does code for proteins that are responsible for many human functions that are vital to life. Just as there are many disorders associated with mutations in nuclear DNA, there is an ever-increasing list of disorders due to mutations in mt-DNA. You will find the future discussion of mt-DNA fascinating, but for now, let’s remember that the mitochondria in the zygote all come from Mom’s egg cell, because the sperm mitochondria are located in its tail, which, as you know, is lost during fertilization. So, any mitochondrial DNA problems in the child have to come from the mother. AND the severity of the symptoms depend upon how many “mutated” mitochondria (those with “bad” DNA) are passed on to the offspring.
3. Base pairing.
Chemically, there are two styles of bases. Purines consist of a heterocyclic pentamer (2 nitrogens, 3 carbons), and a heterocyclic hexamer (2 nitrogens, 4 carbons), joined to each other. Two of the carbons are shared by both the pentamer and hexamer ring. Adenine and Guanine are purines.
The following is the general structure of a purine:

Pyrimidines consist of a single heterocyclic hexamer (2 nitrogens, 4 carbons) ring. Cytosine and Thymine are pyrimidines. The following happens to be cytosine:

The following diagram illustrates the four nucleotides in DNA. A nucleotide is composed of one of the bases, a pentose sugar called deoxyribose, and a phosphate group.

The classic chemical bond between purines and pyrimidines is normally Adenine to Thymine, and Guanine to Cytosine. They are joined by hydrogen bonds. Notice that there are two hydrogen bonds between Adenine and Thymine, and three between Guanine and Cytosine.

The stereochemistry (physical geometry) of the whole molecule makes this a necessity, since a purine-purine bond would be too big to fit into the double helix, and a pyrimidine-pyrimidine bond would be too small. Also, hydrogen bonds are strong enough to keep things together, but not so strong as to prevent the separation of one helix from its sister. The stability of the bonding system is further enhanced by the formation of hydrophobic bonds between the next adjacent base pair, either above or below it. This is called base-stacking. Although hydrophobic attractions are not really chemical bonds per se, they keep the molecule stable but flexible, and allow for its general shape to stay true.
Having said all that, when we move onto the other major nucleic acid, RNA, there are some variations. Instead of Thymine, RNA contains the pyrimidine Uracil. Uracil pairs with Adenine in RNA. Thymine and Uracil are both pyrimidines. Thymine is methylated (CH3 or H3C) and Uracil is not, as shown below:

It is amazing how much we know.

The classic DNA double helix — the Watson-Crick model (again, with continued apologies to Rosalind Franklin) — is called the B-form of DNA. This is what you saw in the original X-ray diffraction pattern earlier. This form has a helical diameter of 2.37 nanometers (nm), a rise of 0.34 nm per base pair, and a pitch (one complete turn of the helix) of 3.4 nm. This corresponds to exactly 10 base pairs per complete turn, (34 divided by 3.4). It also allows for the formation of a major groove opposite a minor groove in the molecule. Most of the DNA in our chromosomes is in this form, but because of the structural flexibility of the molecule, and the way it is packed in the chromosome, other forms of DNA exist consistently in different parts of our genome.
The A-form of the molecule is a little more “squished”, with a 0.29 nm rise in base pairs, and a 3.2 nm pitch.
The Z-form is stretched out to the point where it is actually flat, much like taking a spring and laying a cement block on top of it. It has a 0.37 nm rise and a 4.5 nm pitch.

So, how do these three forms of DNA perform as far as transcription is concerned? Whoa! Haven’t defined those terms yet!!! Well, whatever they are (coming shortly), the definitions of transcription and translation do not change from one form to the other. The wider the major groove, the less chance there will be for errors in reading the genetic code. Narrow, flat, or otherwise mis-shapen grooves in DNA can sometimes obscure nucleotides, so B-DNA with wide, deep major grooves, will give the most accurate reading, while the other two forms are more open to mutations caused by misreading.
OK, I hear the sigh of relief. Darn good thing I don’t have to commit all this to memory for an exam. I hear you – really!
Anyway, here is a picture of the three different DNA forms — B-DNA is on the left; A-DNA is in the center; Z-DNA is on the right.

5. DNA replication.
Every time a cell divides, DNA replicates itself. For many years, it was thought that the molecule simply unzipped, producing two helices. Each helix then built a complement, from one end to the other, upon the two “scaffolds”. This produced two identical molecules. Certainly, replication does produce two identical molecules, but the replication process is not as simple as that. We now know that there are three phases of DNA replication:
• Initiation involves enzymatic recognition of the place where DNA replication begins.
• Elongation results in the creation of a “replication fork”, upon which the new polynucleotide strand is produced.
• Termination occurs after the two new double helices are produced.
Initiation regions have been identified in yeast, E. coli, and other simple organisms that have been genetic tools for many years. In yeast, the initiation of DNA replication begins at a 200 base pair sequence called the Autonomously Replicating Sequence (ARS), and an adjacent 40 base pair Origin Recognition Sequence (ORS). The ORS provides a binding site for a set of six proteins, called the Origin Recognition Complex (ORC). The ORC binds, and then attaches to the ARS, and replication is initiated. The human genome contains regions very similar in nature to the ORC, as evidenced by proteins produced from this gene. As a result of initiation, replication forks are produced. A replication fork is simply a place where the DNA unwinds — the two arms of the unwinding form a fork.

Elongation occurs in a very precise manner. Replication occurs on one strand (helix) in a 5′ to 3′ direction in a continuous manner. This is called the leading strand. Replication occurs in a discontinuous manner in the 5′ to 3′ direction on the other helix called the lagging strand, but in segments called Okazaki fragments. The diagram below shows a replication fork and the two different kinds of polynucleotide addition:

OK, what is all this 5’, 3’ hocus-pocus? The 3’ (3-prime) end of the molecule is the hydroxyl end. The DNA polymerase (appropriately named) needed to synthesize new strands is very fussy, as most enzymes are. It can only attach a new nucleotide to the 3′ (-OH) group using a typical phosphodiester bond. The “black” 3’ end is where the enzyme binds, and the antiparallel DNA strand (pink) replicates continuously. The 5′ end has the fifth carbon in the ribose sugar and its end. Since the polymerase can only replicate in a 3’ to 5’ direction, it must do it one piece at a time on the other strand, causing a lag. These pieces are the Okazaki fragments.
Now I would like you to view a really nice animation of the full process. As I am sure you aware, any molecular dynamic like this involves enzymes. The captions on the following animation talk about the enzymes that are involved in the process, and animates replication. FAIR WARNING: Click on the link and look at it only when it is quiet, and you have tons of patience. I guarantee you will have to view this several times before the concept of replication becomes clear to you —
Replication animation
( http://www.johnkyrk.com/DNAreplication.html )

Wow! This is a much more involved process than the simple, but now outdated and incorrect, “unzipping” of the molecule.
Incidentally, the lower left of this link you just looked at has some “DNA Anatomy”, “transcription”, and “translation” animations. You have already seen similar animations above, but these are different pictures, different colors, etc. They may or may not be helpful, but you can’t learn from looking at them only once – multiple visualizations are essential.
Now that you have an idea of how DNA replicates, let’s talk about its MOST IMPORTANT, and interestingly enough, its ONLY function. Go on to the next section.
( Topic 2 ) The incredible “GRANDE” summary of peptide synthesis
For all that we have heard about DNA, it has ONLY one function: to be sure that the correct amino acids are in the right sequence in a peptide. That’s it – that’s all it does! And, want to know what else? There are two steps to accomplish the proper synthesis of a peptide, and DNA is involved in only the first one of these. Seems like this much ballyhooed molecule doesn’t do a whole lot.
By the way, for all of you that are chemistry purists – please congratulate me on the proper use of the term “peptide” and not “protein”. Just to review, a “peptide” is simply a short sequence of amino acids. Generally, the standard says that if it is 50 or less amino acids, it is called a peptide. Most of our proteins are much longer than that. And, they are synthesized as the result of many genes making its own peptide. Joining those peptides together into a protein is also an amazingly elegant process. Later in the course, we will have a section on proteins, when I will introduce you to some proteins that actually have a role of joining together those peptides. These are called CHAPERONES. What? That’s right – tune in later in the course . . . . . . .
As we said above, there are TWO steps in building a peptide:
(1) the first step is called transcription: the end-product of transcription is the synthesis of a molecule called messenger RNA (hereafter referred to as “mRNA”).
(2) the second step doesn’t even involve DNA, but let’s include it anyway; this is called translation: the synthesis of amino acids into a peptide.
In the first module, we reviewed the incredibly fascinating DNA molecule. Now we will use this section to assemble that info into a unit on the molecular dynamics of peptide synthesis. Some of what follows includes a review of concepts covered in the previous section — some of it is new. So, be sure your body is well-anchored as we delve into all of the details. Just one more point before we get our feet wet: the previous unit did not include anything on RNA. We will include in this section the “classic” view of RNA. However, in the last decade or two, we have found out that RNA has many forms and multiple functions. These are so numerous, that we are setting aside a separate unit just to sing the praises of RNA versatility. That will be in the next section, but first:
As we just stated, the definition of transcription is straight-forward: one of the helices of DNA is used as a template (“pattern”) for the formation of RNA. BUT – – – – – –
When we look back at the historical (oftentimes hysterical past) it was always thought that the nucleotides making up messenger RNA represented the exact complement of the DNA from which it was derived. So, if the DNA pattern was 1000 nucleotides long, the mRNA derived from it would be 1000 nucleotides long, and exactly complementary to its mother DNA. WRONG!!!!!
About three decades ago, this “gospel” about messenger RNA started to come into question. At that time, as technology became more sophisticated, it was very clear that mRNA exiting a cell nucleus was made up of only a VERY small fraction of the equivalent bases in DNA that it came from. This fraction, in some cases, was estimated to be as low as 3 – 5%. We now know it can be as low as 1%. So, if the nuclear DNA of interest was 1000 nucleotides long, the messenger RNA that resulted from that, and exited the nucleus, could be 10 – 50 nucleotides in length, or less!!!

PAUSE: Before you go on, please re-read the previous paragraph enough times so that you really digest it. This forms the basis of what is to come.
You good? READ IT AGAIN (PLEASE?) OK ——-
After many studies, all of science was astounded by the revelation that most of our nuclear DNA does not even transcribe!!! This is why the mRNA was so “short”. In fact, experts currently believe that well over 95% of our DNA does not code for protein!!! For this reason, the term “junk DNA” was coined for this, apparently non-functional, fraction of the molecule. It is now very clear that there are concrete functions for most of the “junk”; but, the term is still being used anyway. It does continue to cause a lot of head-scratching among the top experts. There are areas of DNA about which we have no idea of function. So, what is going on?
The following article, published about three months ago, describes the new thinking about DNA that does not code for proteins. It really explains well the new thinking about the so-called “Conserved Non-Coding Sequences” (CNS’s). You will definitely need some quiet time to digest this, so if that is the case now, click, read, and enjoy:
It is not “junk”after all!!
http://www.biologynews.net/archives/2014/03/31/new_functions_for_junk_dna.html

Before you go on, we need to avoid a bit of confusion. The terms “initiation”, “elongation”, and “termination” are used to describe BOTH replication – covered in the last section – and transcription – about to be covered now. The terms have VERY DIFFERENT meanings in each of the two processes. So, you can either do something easy, or something hard:
(1) Easy — ignore those terms from the last section, and pretend you have never heard them before as you go through transcription,
(2) Hard — remember what they mean in replication, and compare and contrast.
Grande summary of transcription – the formation of messenger RNA
(1) Recall that there is a one-to-one relationship between DNA bases and RNA bases when RNA is built on a DNA template.
(2) The pairing relationship when DNA forms RNA is G with C, and A with U. This RNA was once thought to be the messenger-RNA that exits the nucleus.
NOT!!!! NO!!!! NADA!!!! NEIN!!!.
The one-to-one base complex is actually called the pre-messenger-RNA (pre-mRNA), or the primary transcript. When this pre-mRNA breaks from its template DNA, enzymes “cleave out” various segments of it. These cleaved-out segments are at different parts on the pre-mRNA, rather than being one continuous sequence. These RNA segments are called INTRONS, and the DNA they come from is called INTRONIC DNA.

A sequence of “GU” is at one end of the intron, and a sequence of “AG” defines the other end. Since this is where splicing takes place, the “GU” and “AG” are known as splice sites. The remaining RNA segments are “spliced together” by other enzymes. These remaining RNA segments are referred to as EXONS, and the DNA they come from is called EXONIC, or CODING, DNA. These spliced-together exons form mRNA, which then exits the cell nucleus and begins the process of translation. Here is a scheme showing how it works:

The big question is this: human beings are very conservative organisms. There is very little anatomy or physiology that is “wasted”. This axiom is certainly defied when this very intriguing RNA “sculpturing” activity is examined. With this in mind, let us proceed:
1. “Initiation” begins with one of two isoforms of an enzyme called RNAP. The acronym stands for (DNA-dependent) RNA polymerase. (The parentheses indicate that sometimes the phrase is not used to name the enzyme). Either RNAP I or RNAP III binds to the 5′ (phosphate) end of the promotor site on template DNA, which serves as the template for what ultimately will become messenger RNA. The DNA sequence that forms part of the 5′ promotor is usually either a “CAT box” (CCAAT) or a “TATA box” (TATA). The RNA complement immediately begins to be synthesized. Given the 5′ promotor on template DNA, the first base in the RNA synthesis will generally be a purine (either Guanine or Adenine).
2. “Elongation” follows when RNAP I or III moves along the template DNA strand and adds RNA purines or pyrimidines complementary to the template DNA. The difference between the two RNA polymerases is merely the rate of synthesis. RNAP I polymerizes at the rate of 20 nucleotides per minute, while RNAP III polymerizes at the rate of 2000 nucleotides per minute.
3. “Termination” happens when the enzyme encounters a termination triplet, or “stop” triplet. In DNA, these would be ATC, ATT, or ACT. This would make the termination triplets in RNA either UAG, UAA, or UGA respectively. When the RNAP encounters any of the triplets, the synthesis of the peptide is terminated.

4. “Capping” occurs when another isoform of RNAP, RNAP II, “caps” the final product with poly-adenine, a process appropriately called polyadenylation. The ultimate RNA that forms, you remember, we called the “primary transcript”, or pre-mRNA.
5. Endonuclease kinase (EK) enzymes then begin to splice out introns from the pre-mRNA. The vast majority of splice sites are 5′-GU-3′, or 5′-AG-3′. So, the EK binds these sites, and a fission of the introns results. Biochemically, the fission occurs when a hydroxyl group attaches to the 2′-carbon. resulting in a cleavage of the phosphodiester bond at this 5′ splice site. The cleaved area then attaches downstream to the cleaved 3′ splice site. This results in the intronic RNA forming a loop, or lariat, at the area. This has actually been visualized using scanning tunnel microscopy. That is, RNA can be viewed with a series of looping lariats on it — all of the lariats are introns that will eventually be spliced out.
6. The lariats are then removed via the activity of these EK enzymes, leaving the remaining exonic RNA fragments.
7. A series of enzymatic complexes are then formed to splice the exons together into the final messenger RNA. Part of these complexes are called snRNP’s or “snurps”. These single nucleotide ribonucleoproteins bind specifically to the “exposed” portions of the exons, and by simple dehydration synthesis, the exons are spliced together. The entire complex of snRNP’s that splice out the intronic RNA is called a spliceosome.
To see how spliceosomes work, go to the web site indicated by the following link, and click on “how spliceosomes process RNA”.
Be sure your sound system is turned on.
Spliceosomes
http://highered.mheducation.com/sites/0072437316/student_view0/chapter15/animations.html

The grande summary of translation – the synthesis of a peptide
Remember that the genetic code (the series of RNA codons that specify amino acids) is universal — the same in all eukaryotic cells. There is no “punctuation” (each nucleotide is read), there is no overlapping, but there is redundancy.
1. The messenger RNA (the series of spliced exons) exits the nucleus via the nuclear pores. It may or may not be golgi-assisted. If not, the molecule is actually brought to the pores via an exocytotic vesicle.

2. Cytosolic polyribosomes dissociate into their two subunits: a 60 S subunit and a 40S subunit. This “exposes” the poly-T m-RNA binding site on an area of the 40S subunit. A specific enzyme prevents the reassociation of the ribosomes so that the binding site remains exposed.

3. The capped (poly-A) end of the messenger RNA attaches to the 40S subunit binding site.
4. Elsewhere in the cytosol, an amino acid connects to the acceptor portion of transfer RNA, which is now called the D-Arm of the molecule. Catalysis of the amino acid joining to the D-Arm of tRNA is done by the enzyme ATS (aminoacyl-tRNA synthetase).
5. The first aminoacyl-tRNA that enters the picture is met-tRNA (t-RNA bound to the amino acid methionine). This binds to an area on the 40S subunit in the neighborhood of the mRNA called the P-site. The specific anti-codon on met-tRNA is UAC. This then “scans” (moves along) the mRNA until it encounters the “start codon” of AUG. The combination of codon and anti-codon takes places via simple complementary base pairing. This area obviously may not be at the very beginning of the mRNA, and it usually isn’t. The area where the met-tRNA binds to the start codon on mRNA is part of what is termed the “initiation complex”.
6. Depending on the next triplet in mRNA, the next aminoacyl t-RNA joins to the next triplet (by base pairing) on a ribosomal area called the A-site. The enzyme peptidase catalyzes the addition of methionine to the juxtaposed amino acid forming a dipeptide. Enzymes called releasing factors hydrolyze the bond between the t-RNA and the P-site. The t-RNA then is removed from the A-site, and joins to a third site on the ribosome called the E-site. The enzyme translocase moves the growing peptide to the A-site as the previous aminoacyl t-RNA binds to the E-site, from which it eventually is removed. Peptide synthesis continues in this manner until a “stop codon” is encountered (UUA, UAG, or UGA).
7. Once peptide synthesis is finished, other enzymes reunite the two subunits of the ribosomes.
OK, so you need a visual to summarize all of this. To view AN INCREDIBLY, REALLY AWESOME (THAT MEANS GOOD) AMAZINGLY EXCELLENT animation of translation, go to the following site, and click on “Protein synthesis”. Be sure to have your sound system turned on, since there is dialogue to accompany the animation. I would suggest that you play it through completely first. Then, go through it again, and click on “pause” periodically to digest each individual step in the process. If you don’t get peptide synthesis at this point, it will become really clear after you go through this animation (they call it “protein synthesis”, but we know the correct term is really “peptide”):
Translation animation
http://highered.mheducation.com/sites/0072437316/student_view0/chapter15/animations.html

This gives you a good picture of the entire process.
(Topic 3) Something special about RNA
In the last section, we looked at the classic role that RNA plays. We have already stated that, for decades, RNA molecules were “soldiers” taking orders from DNA and carrying its message to the cytoplasm so that a peptide could be synthesized. The one thing NOT often covered in textbooks, involves a different role for RNA. It is now clear that certain small RNAs might actually direct nuclear genes to turn on or off during development (What??? RNA is telling DNA what to do? – YUP). This process orchestrates tissue differentiation, organ system development, and proper embryogenesis. So, let us address that now. In what has been one of the hottest research areas in the last few years, Dr. Craig Mello (of the University of Massachusetts), won the prestigious Nobel Prize for the work he pioneered on a topic called “RNA interference”.

RNA interference silences genes
1. VERY short RNA fragments, 21 to 28 nucleotides in length, have been observed for many years. However, because of their much larger relatives, they were ignored as being innocuous, or simply discarded when they turned up in research venues.
2. Early in 1990, agricultural geneticists discovered, quite by accident, that such small RNAs could suppress the expression of various genes in petunias. Appropriately, this process was named “cosuppression”. Later, this same phenomenon was observed in animal cells. Dr. Mello performed an experiment where RNA oligonucleotides were delivered into worms, but they used double-stranded molecules, which are really single stranded RNA that “kinks”. However, these are organized kinks, in that there is complementary pairing, giving a double-stranded appearance. The results were rather unexpected — this RNA quelled stretches of DNA, which, as you know, helps generate the RNA in the first place.
3. No, this is not a Catch-22. This phenomenon, which was earlier seen in petunias, and later seen in flies and other organisms, is known as RNA interference, or RNAi. So, we now know that RNA can control DNA development by inhibiting its expression. Talk about “biting the hand that feeds you”!!!
4. The whole process starts with antisense RNA. This is RNA that is synthesized complementary to mRNA (the usual stuff transcribed from a gene of interest).
a. The antisense RNA and mRNA hybridize, and produced a double stranded RNA duplex called dsRNA.

b. An RNAse III endonuclease enzyme, appropriately called “dicer”, cleaves this heteroduplex into small strands of RNA called siRNA (the “si” stands for “small interfering”). This, obviously, “interferes” with the activity of mRNA attempting to make a protein. In fact, using genetically altered strains of the roundworm C. elegans, scientists in fall of 2000, discovered that this process of dsRNA formation is actually responsible for genes that cause the DEGRADATION of mRNA (ouch).
c. A compound called “RISC” (RNA-Induced Silencing Complex) has a sequence of RNA complementary to the one being cleaved. Once the complementary pairing has occurred, the endonuclease then cleaves the target RNA.
d. Remember that this renegade antisense RNA has to be manufactured by GENES!!! So, some of these genes that are responsible for this RNA interference appear to also be involved in nonsense-mediated decay, a protective mechanism that may be used by cells to proofread newly created messenger RNA (mRNA) and to prevent the production of defective protein molecules. That’s right!!! I’ll bet that you were thinking that destroying mRNA was a bad thing – it isn’t if only the mRNA that makes defective proteins is destroyed, and the “good” mRNA is left alone. In fact, when we talk about the genetics of cancer, we will mention this RNA interference as a possible preventative at the molecular level.
As a way of gathering more data about this very unique process, molecular biologists have used double-stranded RNA to degrade mRNA in cells to shut down the effects of specific genes. and to find out what is involved in the selection. Of course, experimental organisms, such as C. elegans, Drosophila and many other cell-types were used. In an amazingly demonstrative experiment, scientists isolated the mRNA responsible for the production of myosin. They then combined the myosin mRNA with its antisense complement, to produce at dsRNA duplex. This was injected into C. elegans. Normal worms became paralyzed, due to the lack of myosin production!!! Therefore the RNA technique completely interfered with normal muscle development by stopping the action of myosin mRNA. So, it works! Of course, this can’t be tried in people, but the evidence is incontrovertible about RNA interference.

OK – here is an illustration of gene silencing. It is a COMPLEX photo, so be sure you have lots of time and patience when you look at this. If patience is not something you have right now, close it out, and come back when your brain is in a fresher state. Ready?

The introduction of dsRNA mimics a gene knockout of almost any known gene by in effect deletion of the mRNA of the gene. The application of RNA interference in mammalian cells has the potential to revolutionize the field of functional genomics. The ability to simply, effectively, and specifically down-regulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential. The old way was to delete a gene to make a knockout, a very time-consuming, error-prone process. Now it is much easier and much more accurate. Studying disease genes will be made easier using RNAi to create knockouts.
RNAi therapy for human diseases
For many years now, gene therapy has been practiced yielding everything from miraculous success, to mediocre results, and to disastrous consequences. The molecule used for this therapy has always been DNA. Now, the potential to eliminate the biological disasters (the most prominent of which will be addressed later in this course) emerges with a new delivery vector: RNAi.

OK, gang, it is now time to read a .pdf article. It addresses both the science and the therapeutic value of RNAi. We have gone over the science, so the only part of the article I have given you summarizes the therapeutic value of this incredible molecule.
When you click on the following link, a .pdf file will open, and you will read an article that extols the virtues of RNAi as a therapeutic paradigm. I have marked the section of the article that I want you to read:
RNA interference — therapy apps.pdf
(Attached)

( Topic 4) Molecular Genetics — you’ll be amazed at the definition of “the basics”
What’s this doing here??? Isn’t something labeled “basics” supposed to be the FIRST thing in a series of lessons?
Not in this case – even though this says “the basics”, I did NOT put it in the wrong place. You will soon see that the following material that I call “the basics” requires AND UTILIZES the incredible knowledge you have just gained about nucleic acid dynamics. OK – now that I have established that this was not put at the end because of a senior moment, let’s look at how this amazing molecule is sliced and diced (well, we have already seen “dicing”), cut, engineered, and custom-built. This will also start to give you an insight into the “Clinical Applications of Molecular Genetics”.
The goal of this section is to build ourselves a technology dictionary. So, yes, we are going to be DEFINING TERMS in the bulk of this section. These terms will be used/applied as we proceed through any parts of the course involving DNA analysis (which will be most of the weeks!!!). As you will see, basic molecular genetic technology has its own unique lexicon, most of which has to do with manipulating, analyzing, and drawing conclusions from DNA. The ultimate goal of this is to deliver accurate information to people about their genomic makeup, so they can make more informed medical and clinical decisions. What follows is (what is known today as) BASIC genetic terminology. These are the most simple, foundational terms, upon which the more complex molecular genetic protocols are built.
Are you seat belts fastened? If not, “click it or ticket”.
Here we go:
(1) Recombinant DNA (r-DNA), as the name implies, is a single molecule of DNA taken from two or more different sources that have been combined using the proper enzymes. The incredible thing about r-DNA is that the sources do not matter. r-DNA can be made by combining plant and animal DNA, human and bacterial DNA, yeast and bacterial DNA, etc. So, if you want a bacterium to make human insulin, the DNA sequence for insulin can be isolated from humans, and “spliced” into bacterial DNA. Because bacteria reproduce so quickly, and their biproducts can be isolated so easily, this becomes a rapid, inexpensive way to synthesize insulin. So, does this mean that if I combine gorilla DNA with mink DNA, I will get a gorgeous coat where the sleeves are too long? No, what it means is that DNA is DNA no matter where it lives, and combinations between almost any two sources can easily be done.
(2) A restriction endonuclease, more generically called a restriction enzyme, cleaves DNA molecules consistently at specific points. These enzymes were first discovered almost by accident. I am not sure of the venue where this happened, but a simple bacterium in culture (let’s call it #1) was contaminated by another bacterium (call it #2). Normally, when a bacterial culture is contaminated, the “invader” takes over the whole container. In this case, the invader was destroyed. Further research demonstrated that enzymes from #1 was able to restrict the growth of #2. It was eventually found that the growth restriction was caused by the restriction endonuclease of one bacteria cleaving the DNA of another.
Further investigation revealed the existence of a whole slew of these enzymes. AND, what is really awesome, is that any given restriction enzyme always cleaved DNA at the same place. The places they cleave are called restriction sites. The resulting DNA molecules are called restriction fragments. A VERY interesting item about restriction sites, is that they are usually “palindromic”, such as A-C-T-A-G-T. In this case, “palindrome” means that the sequence reads the same from 3′ to 5′ on one helix, as it does from 5′ to 3′ on the other helix. So if you write the complement to the given sequence, it reads T-G-A-T-C-A, which is palindromic to A-C-T-A-G-T. Because there are so many restriction enzymes, each cleaving at a specific place, DNA molecules can be “custom-cut” with the right “cocktail” of enzymes. When they were first discovered, the term “DNA scissors” was used to describe their activity. Apropos at that time, but the term is not used any more. This picture shows more specifically how and where these enzymes cut DNA – note the G-A-A-T-T-C palindrome:

From this information, we can construct another term which you may have heard before. Restriction enzymes cleave at palindromic restriction sites (as above). Different DNA sequences, such as in a normal and disease gene, have different palindromes. So, using the same restriction enzymes will cleave both the disease and the normal gene in different places. This means that when the genes are cleaved, the restriction fragments will have different lengths. In other words, you have a length polymorphism. So, a restriction fragment length polymorphism, or RFLP, when properly analyzed (LATER!!) can determine the difference between a disease gene and a normal gene.

Because there are so many restriction enzymes, nomenclature has to be consistent and specific. So, each enzyme is named according to the bacterium from which it is isolated. Using the first letter of the genus name, the first two letters of the species name, and a number indicating the strain or type of bacteria, the enzyme name is constructed. The enzyme isolated from Bacillus subtilis, strain 3, is named Bsu3. Escherichia coli, strain 2, enzyme is named Eco2. Streptomyces cerevisiae, strain 1, enzyme is named Sce1, etc.
(3) The polymerase chain reaction, or PCR, is a technique for taking a segment of DNA and making copies of it in geometric multiples…1, 2, 4, 8, 16, 32, etc. This process is referred to as DNA amplification. A machine called a thermal cycler has the ability to produce multiple copies of DNA by continuously doubling the number of products. Thus, with only 20 cycles, you generate 220, or over 1,000,000, copies of the original (what??? Yes, do the math: 220 does equal more than one million!!!).
Here is a summary of the lab protocol:
a. The DNA segment containing the sequence of interest is first denatured — that is, the two members of the double helix are separated.
b. An oligonucleotide primer, a short segment of DNA, is then added to each helix. The enzyme DNA polymerase catalyzes the addition of complementary nucleotides to each helix. However, the denaturing and addition of nucleotides, and repetitive continuity of this process, must be done under high temperature. Since enzymes normally denature under this kind of heat, one kind of the polymerase used is taken from thermophilic bacteria, such as Thermus aquaticus, which normally lives in hot springs. So, the enzyme is called Taq DNA polymerase.
The following is a down-to-earth, but still amazingly cool, animation of how PCR works. It illustrates the process well using animation, and you can pause, stop, go back, review, at whatever pace allows you to learn the process. But again, you need quiet and patience to really digest this. Click on the following link and watch the geometric multiplication of a “target” DNA:
The Polymerase Chain Reaction
(http://www.sumanasinc.com/webcontent/animations/content/pcr.html )

(4) Cloning is a term you have recently heard in the news a lot. A sheep named Dolly was cloned in 1997 (by the way, do you know why she was named “Dolly”? Because she was cloned from a mammary gland cell – think about it – it will hit you). A plethora of other mammals have since been cloned using the same technology.
Cloning at the molecular level has quite a different meaning. Using the right cocktail of restriction enzymes, a DNA sequence of interest can be cleaved out of larger DNA molecules. Many copies of this can be made by PCR amplification. These restriction fragments are then integrated into bacterial DNA (because recombinant DNA is easy to make), and are appropriately called inserts. For one point extra credit on this week’s assignment, which bacterium would you use, and why? The bacterial “host” for the insert is called a vector. What is nice about most bacteria, is that the inserts do not have to be placed in the main circular chromosome, where they might disrupt normal bacterial function. Rather, most bacteria have smaller, endoplasmic, circular chromosomes called plasmids, which can be genetically fitted with inserts without disrupting bacterial replication. When these bacterial vectors reproduce, they also reproduce, or clone, the DNA of interest.

(5) A library is a collection of recombinant clones. The library contains a multitude of bacteria, each containing a particular sequence of DNA (like for insulin). So, if I want to investigate a sequence of DNA, I can “check out” a bacterial culture that contains DNA for a single protein or a single polypeptide. Or, I can check out a culture that contains DNA that causes a particular disease. You can see that the uses of these libraries is quite ubiquitous.

(6) Complementary DNA (c-DNA) is a sequence of DNA that is the exact complement of a messenger RNA molecule. Remember that m-RNA is a combination of exons — all of the intronic RNA has been taken out. So, the best thing about cDNA is that it represents only the portion of DNA that codes for a particular protein. To make a cDNA molecule, m-RNA has to be isolated from the polyribosomes in the cell cytoplasm. Using sophisticated lab technology, and an enzyme called RNA-directed DNA synthetase, a DNA complement to the m-RNA is constructed. Look at the name of that enzyme again. Normally, DNA directs the synthesis of RNA — this is how transcription works. However, this enzyme reverses the process, and uses RNA as a template to make DNA. Therefore, this enzyme is known by its more familiar name — reverse transcriptase. And yes, the newly synthesized cDNA can be introduced into a bacterial vector, creating a cDNA library.

(7) A probe is a device used to find something. That’s what it says in any good dictionary, and that’s what a gene probe is. Actually, the best probes are cDNA molecules that have a radioactive tag. If we want to determine if a particular sequence of DNA exists in a cell nucleus, we can make cDNA, tag it with radioactive phosphorus (P32), and introduce it into a nucleus. Using radioactive tracing equipment, we can determine if the cDNA has hybridized to its complement (or not). Because this DNA is probing much larger territory, the name is certainly appropriate.
(8) Southern blotting is an old technique (almost 50 years old). However, this technique is still used a lot for identifying, categorizing, and classifying segments of DNA. First, DNA is isolated, and “blasted apart” into random-sized fragments by a large number of restriction enzymes. Next, these restriction fragments are subjected to gel electrophoresis. The purpose of this process is to allow all the fragments to travel through a gel column, with the largest fragments staying near the origin, and the smaller ones, further away. The gel is then blotted onto a nitrocellulose or nylon filter to make the fragments easier to handle. A cDNA probe with a radioactive tag is then washed across the filter. The filter is exposed to X-ray. If the DNA of interest is present, the cDNA will hybridize to it on the filter, and the X-ray will reveal its presence as a dark band. If not, no dark band.
Click on the following link to see a diagramatic representation of Southern Blotting:
Southern Blotting

(9) Northern blotting uses the same technology as Southern blotting, but to detect RNA.

(10) Western blotting uses the same technology as the other two, but to detect either whole proteins, or polypeptides.

By the way, you may have wondered where the names for the blotting techniques originated. Southern was actually the last name of the person who developed Southern blotting. Because a number of geneticists have weird senses of humor, the techniques for RNA and protein were given directional names. Aren’t you glad you asked?
(11) Last, but not least, is the term single nucleotide polymorphism. Just looking at the term, you can guess that this means a change in one nucleotide. Single nucleotide changes can wreak disaster on the person, creating any number of diseases. Given that there are 3 billion nucleotides in the genome, the potential for these changes is enormous. And by the way, we can use the acronym to refer to these changes; the acronym being SNP. Yes, you can pronounce it “SNIP”.
The DNA microarray chip and expression profiling
Over the last 20 years, a technology has been developed to aid with visualizing, sequencing, and detecting changes in DNA. Over the past dozen or so years, this technique has been modified to allow us to assess and evaluate the expression of mRNA. These tools are called DNA microarrays, also called DNA arrays or “gene chips”. DNA microarray chips come in many varieties. Whether they are created by scientists or produced commercially by one of several companies, arrays depend on the same basic principle. Let us pause now and review some of these basics. Here goes:

The basic principle is absolutely second nature to us all by now (it better be!): complementary sequences of nucleotides hybridize with each other. For example, a DNA molecule with the sequence “A-T-T-G-C” will hybridize to another with the sequence “T-A-A-C-G” to form a double-stranded DNA oligonucleotide sequence. Using Southern Blotting, as you remember, single-stranded DNA with a known nucleotide sequence is labeled with a radioactive isotope or fluorescent dye and then used as a probe to detect a fragment of DNA. Autoradiographic analysis shows us whether or not a particular DNA sequence, or even a whole gene, exists in the sample of DNA being analyzed. We did not, though, talk about the use of this procedure for mRNA expression. So, let us summarize that a bit here. We’ll find that probing for RNA is pretty much the same as probing for DNA.
Gene expression
If one of our genes, let’s call it gene “XYZ”, is still “switched on”, it will make a primary transcript, which ultimately becomes mRNA. Otherwise, it will not. Therefore, mRNA is said to be the expression of the original DNA – therefore, the term gene expression. To verify that XYZ is expressed, you make a radio-labeled DNA probe from as large a DNA oligonucleotide segment of “XYZ” as your equipment can handle. Then, you isolate mRNA from the tissue. The technology does exist to “catch it” as it exits the nucleus!!! You then bind that mRNA to a solid medium (such as a nylon filter), and then wash the “XYZ”-DNA probe over the filter. If gene “XYZ” is expressed in the tissue, that means mRNA has been synthesized, and you will see the radioactive signal on your autorad. Just to be different from Southern Blot, this procedure is known as a Northern blot. The following Northern blot is easy to interpret: the heaviest fragment, 5450 nucleotides is the primary transcript. As more and more introns are spliced out, it gets lighter and lighter, until the number of exonic bases = 1100. Since this is mRNA, you will also find it in the cytoplasm of the cell, where it does its thing.

Just as an aside, with all of our high tech prowess, this cannot be done without paper towels — YEAH, Bounty!). Anyway, would it not be wonderful if we could do thousands and thousands of these Northern Blots all at once in the same place? That way, we could get a quick idea of how many of a person’s genes are being expressed (making mRNA) all at once. I don’t think it is possible for you to write fast enough what you would be able to do with this information. Disease diagnosis, family studies, forensics, clinical analysis, and on and on. Imagine the power of being able to do thousands of these experiments at a time. Yup! That’s what the DNA microarrays do. For now, the headline version of this is that DNA probe detection is done (paradoxically!) on a much larger scale with a microchip (that sounds like an oxymoron, like “jumbo shrimp”). Instead of detecting one gene or one mRNA at a time, microarrays allow thousands of specific DNA or RNA sequences to be detected simultaneously on a glass or plastic slide about 1.5 centimeters square (about the size of your thumb).
Each microarray is made up of many bits of single-stranded DNA fragments, or RNA fragments, arranged in an “x” by “y” grid pattern laid out on the glass or plastic surface. What did he say? Think about this mind-boggling feat — “marrying” nucleic acid molecules to computer chips. HOLY COW! When sample DNA or RNA is applied to the array, any sequences in the sample that finds a match will bind to a specific spot on the array. A computer then determines the amount of sample bound to each spot on the microarray. Further, if the applied DNA is labeled with a colored fluorescent tag, it can also be visualized. So, on your “x” by “y” microarray chip, you can see colored signals in each cell of the array if hybridization has occurred. You can then visually see if a gene of interest in your tissue sample has been expressed. Is that COOL or what???
Expression profiling
Another way to use this technology is to convert RNA to complementary DNA (cDNA), which is easier to work with than RNA, and tag it with a fluorescent label.
1. In a typical microarray experiment, cDNA from one sample (sample A) is labeled, say, with red dye, and cDNA from another (sample B) may be labeled with green dye.
2. The fluorescent red and green cDNA samples are then applied to a microarray that contains DNA fragments corresponding to thousands of genes.
3. If a DNA sequence is present both on the array and on one or both samples, the sequences bind, and a fluorescent signal binds to a specific spot on the array. These signals are picked up using a reader or scanner that consists of lasers, a special microscope, and a camera, which work together to create a digital image of the array. Computer software calculates the red to green fluorescence ratio in each spot. The calculated ratio for each spot on the array reflects the relative expression of a given gene in the two samples. Got it? OK, here’s a “for example”:
a. A red signal indicates that a particular gene is expressed in sample A but not sample B.
b. A green signal that the gene is expressed in sample B but not sample A.
c. A yellow signal means that the gene is expressed at roughly equal levels in both samples (because red and green make yellow !!!).
d. No signal means that the gene is not expressed in either sample.
These colored results are known as the gene expression profile or signature. Here is an example of what a very tiny segment of one of these chips look like:

Relook at that Northern blot above. Each one of those colored spots above represents one of those procedures! You can easily see the red, green, and yellow profiles (signals). No color means there is no expression in that particular cell of the array.
Applications of expression profiling
1. If you compare the expression profile of a cell that is in G0 (G-zero) or G1, to the profile of one that is dividing, you can determine which genes are turned on during cell division.
2. If you compare the expression profile of a cancer cell with that of a normal cell, you can actually use microarrays to diagnose different cancers. In fact, specific microarrays have been developed for specific cancers. Breast cancer chips, leukemia chips, colon cancer chips, and others, are currently in use. In fact, you can even distinguish within a particular type of cancer, such as the difference between acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). This cuts down on time and cost, and increases accuracy. NICE!
3. SNP’s can be detected by using genotyping arrays or SNP chips (pronounced “snip chips”). These types of arrays carry all the possible variations of one gene or several genes in a grid pattern. A DNA sample is extracted, multiple copies of the gene or genes of interest are generated using PCR, and the sample is then applied to the chip. The spots that “light up” correspond to the particular gene variants the individual has. By looking at several SNPs at once, researchers can identify SNP signatures associated with a specific disease or a response to a drug. Individuals at risk for a particular disease could then be tested for the unique profile, or signature.
Protein Arrays
Protein chips? Protein arrays? Sure. We have already turned science fiction into science fact, so why not? Microarrays that can be used either to identify and quantify thousands of different proteins at once or to find associations between different kinds of proteins and between proteins and other molecules have been developed, and are currently in use to help out with the human proteome project.

A good example involves arraying antibodies. Antibodies are arrayed on a glass or plastic slide. Each antibody can bind to a different antigen. When a mixture of antigens is applied to the array, such as a mixture of blood antigens, for example, the antigens recognized by the array-bound antibodies will bind to the array. Bound proteins can be detected either by adding a second antibody tagged with a fluorescent molecule or by chemically labeling all the antigens in the blood sample before adding the sample to the array. Each bound antigen-antibody complex can therefore be detected as a signal on the array, and the intensity of the signal roughly represents the amount of antigen in the blood sample. Or, obviously, if it is present or absent. This type of array, like a gene expression array, can be used to generate signatures for different cell types and tissues. It can also be used to determine if complement fixation has taken place — I am not going into that, but, trust me, it can simplify the process of identifying gene mutations in the complement cascade.
OK, now you want to really hear something awesome? You can do the opposite of the above” bind the antigens to the array instead. Are you sitting? A number of groups have shown that these antigens, as well as many other classes of protein, can bind to the glass array, and still retain their biological activity. So, if you want to find out which ligands bind to which protein, wash them across the array, and a signal will show you where the interaction occurs. This would also be good, as you have guessed, to determine protein-protein interaction. Other types of protein arrays are able to detect DNA sequences that bind to proteins or sequences that are chemically modified in the genome, such as by methylation or acetylation. Good grief, Charlie Brown!
Here is an example of a protein microarray. (A) The proteins binding to the epoxide surface are antigenic proteins. (B) a mixture of antibodies are added to the preparation, and (C) green spots and red spots indicated hybridication of antigen-antibody. A blank spot (there are none on this hypothetical diagram) would indicate no hybdridization.

Yes, we haven’t even scratched the surface (well, we actually did that) in trying to develop the applications of this astoundingly remarkable technology.
That is, at least, a partial dictionary of some of the basic terms in Molecular Genetics. Yes, there are many more, but this gives us a start. We will continue to add to our dictionary as the course proceeds.