Daily Newsletter: October 31, 2013 - Meiosis

Daily Newsletter

October 31, 2013 - Meiosis

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In mitosis, we saw nuclear division within a somatic cell . When coupled with cytokinesis, we saw that the goal was taking one parental cell and creating two genetically identical daughter cells. With meiosis, we will again experience nuclear division, but with a different goal than mitosis. The goal of meiosis is to produce gametic (reproductive) cells. As such, we will 'always' couple meiosis with cytokinesis.

Goal of Meiosis: From one diploid parental nuclei, generate four genetically unique haploid nuclei.

Goal of Meiosis-Cytokinesis: From one diploid parental cell, generate four genetically unique haploid cells.

The production of gametic cells requires two nuclear division events: a reduction division, and an equatorial division.
Important Note: All forms of nuclear division have the same basic stages. Prophase is the set-up, Prometaphase removes the nuclear envelope & attachment of mitotic spindles, Metaphase is when chromosomes align, Anaphase is when chromosome/chromatid seperate, and Telophase is when the nucleus returns to "norm".

The first meiotic divison is a reduction division, and is referred to as Meiosis I. As the term reduction implies, we are decreasing something. Specifically, during the reduction division the cell is reducing chromosomal number. REMEMBER: a diploid organism (2N) carries two of each chromosome (one maternal set, and one paternal set). During a reduction division, each set of chromosomes are separated and moved to opposite sides (poles) of the cell.

Homologous Chromosomes: This term refers to the two copies of each chromosome. For example: Chromosome 1 in humans is the largest of the chromosomes. Individuals have two copies of chromosome 1: one from their mother and one from their father. These two individual examples of chromosome 1 are considered homologous (agreeing or consistent structure). Homologous chromosomes carry the same genes, but each homolog (one copy) will have a unique set of alleles (one allele for each gene).  Allele: individual variations in a gene produced by mutations that alters the affect of the gene product.

The image to the right is an example of two different homologous chromosomes. Notice that at the same gene is found at same location on each homolog (a member of a homologous pair). As shown in the first homologous pair, one homolog carries allele A for the first labelled gene, while the second homolog carries allele a (e.g., alleles are individual variations in the genetic code of a gene; this could be a simple SNP for example that changes the function of the gene product, or a more substantial variation that fails to produce a gene product).

IMPORTANT NOTE: Remember that what we are seeing are chomosomes. The two chromatids of a given chromosome are the products of DNA replication. The image to the left should help you remember that you start with a Maternal DNA molecule and a Paternal DNA molecule. After DNA replication, these will consense into maternal and paternal chrmosomes. It is critical that you remember the difference between Chromosome and Chromatid.

Prophase 1 (prophase of Meiosis I) is the most critical stage of meiosis. Prior to the reduction division (which occurs in Anaphase 1), a recombination event will occur between the maternal and paternal chromosomes. Recombination is a genetic event in which homologous genes on two homologs are swapped between the molecules. A section of each DNA molecule is cleaved and then bound to a new molecule of DNA. The resulting molecules will still have the same genes, but the alleles (i.e., variations) they carried have been swapped.

This recombination event begins when homologous chromosomes are brought together during prometaphase I. The homologs possess DNA sequence similarities, and are able to bind to each other (the homologs chemically recognize each other). Regions where Cross-Over (recombination) can occur begin to over lap, forming a visible structure known as a chiasma (pl. chiasmata). During cross-over(recombination), alleles are swapped between the two chromosomes, with the end result being genetically unique allele patterns on each chromosome. CRITICAL POINT: the result is two unique chromosomes! This step is critical for maintaining diversity in diploid (eukaryotic) cell systems. Ever generation inheriets a unique genetic composite of maternal and paternal alleles.

This event is one of the most critical ways that eukaryotic organisms ensure the diversity of their populations. Why do you need to maintain diversity?

After this recombination event, Chromosomes will line up along the equatorial line (metaphase plate) so that there is one homolog on either side of the equatorial line. RECALL: in mitosis, chromosomes lined up so that there was a chromatid on either side of the metaphase plate; now in meiosis I, there is a homolog chromosome on either side. Why is this difference in alignment important?

Metaphase in Mitosis

Meiosis I

As you can see in the image of meiosis I, the alignment of chromosomes during Metaphase is critical. During anaphase I, the freshly recombined homologs are separated. The cell moves from a diploid (having 2 sets of chromosomes, 2N), to a haploid state (having only 1 set of chromosomes, 1N). This is the reduction division!
Mitosis II occurs like mitosis, which is classified as an equitorial division. In an equitorial division, the chromatids are seperated. (see the difference?) NOTICE: each cell produced in meiosis I now undergo meiosis II.

During the reduction division (meiosis I), you went from a diploid to a haploid state. This is done by aligning the chromosomes in metaphase so that there is a homolog on either side of the metaphase plate. During anaphase, the homologs are seperated.

During the equitorial division (meiosis II), each chromosome is seperated into the individual chromatids. In metaphase II, chromosomes are aligned so that the chromatids are on either side of the metaphase plate (or to put it another way, they are alinged down the centromere). During anaphase II, the sister chromatids are then seperated.

The result: 4 genetically unique haploid cells!

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Daily Challenge

We have discussed the main stages of nuclear division, and you have readings from your textbook and supplemental reading on meiosis. In your own words, describe the process of meiosis, complete with a discussion of synapsis, chisamata, and cross-over. Why is the recombination event so critical to population diversity? How could recombination affect evolution?

Daily Newsletter: October 30, 2013 - Somatic Nuclear Division

Daily Newsletter

October 23, 2012 - Somatic Nuclear Division

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Mitosis describes nuclear division of a somatic cell. We commonly use the term loosely to describe cell division, but that is an incorrect usage of the word mitosis. Cell division is better described by the term cytokinesis. It is important to use these terms correctly, as it will help you as you go further through biology.

Mitosis describes a nuclear division event, specifically in a somatic (as opposed to a sexual reproductive) cell. Most of the time we think of nuclear division occuring just prior to cytokinesis (cell division), but there are examples of cells that can undergo nuclear division without undergoing cytokinesis (coenocytic fungal cells have multiple nuclei enclosed by a single cellular membrane). For most eukaryotic cells though, mitosis will be followed by cytokinesis. Remember mitosis and cytokinesis are two different events.

Mitosis is also described as somatic nuclear division. The word somatic refers to general body cells. This is to contrast difference between a general cell and a reproductive cell. A reproductive cell, or gametic cell, will undergo a special type of nuclear division known as meiosis, which will be discussed in Thursdays's newsletter. Gametes are needed for sexual reproduction.

The goal of mitosis is to produce two daughter nuclei that are genetically identical. Coupling mitosis and cytokinesis results in the formation of two genetically identical daughter cells.

Prior to mitosis, DNA replication will have already occured. Every molecule of DNA will have undergone replication (DNA synthesis). We will discuss replication later this week.

The process of mitosis occurs in 4 Main Phases (there are additional phases that have been added in recent years). Below is a phrase to remember:

"Prophase sets up the process, metaphase aligns the chromosomes, anaphase seperates the chromosomes, and telophase returns the cell to normal."

These phrase describes the basic action of the four main steps: Prophase, Metaphase, Anaphase and Telophase.  In the past ten years, prometaphase has been included as a stage to help distinguish events that occur as the cell moves from prophase to metaphase.  You must remember that the stages are artificial attempts to describe a continuous process.  Once the M Phase begins (in this case mitosis), it does not stop until completion or the cell receives a signal for apoptosis.

[Review the University of Arazonia's Mitosis Tutorial. This will take you through the main phases, and includes the Prometaphase addition.]

In Prophase, we see the condensation (packaging) of the chromosomes. We also see the formation of the mitotic spindle. The image to the right shows Early and Late Prophase. In recent years, the term Late Prophase has been renamed as Prometaphase. This was done to indicate the rapid changes that being to take place within the cell, and to indicate the passing of a check point (NOTE: cells go through check (or restriction) points where signals will tell the cell to either proceed or abort the process of nuclear or cellular division. This restriction point deals with whether the mitotic spindle is correctly forming or not (if not, apoptosis).

In Prometaphase, microtubules of the mitotic spindle reach toward the centromere of each chromosome, forming the kinetochore. During this period, we will also see the dissolution of the nuclear envelope. The nuclear lamina (intermediate filaments) and nuclear pores begin to dissociate.

During Metaphase, the chromosomes are aligned down the imaginary equitorial line of the cell. This line is equidistant between the centrioles. The alignment is important. If you look at most drawings, the chromosomes are shown as aligning so that their centromeres are on the equitorial line, and individual chromatids are on either side of the line. This is to represent an equitorial division. As each sister chromatid of a chromosome represents a complete DNA molecule, the division of these will result in an equal number of chromosomes on either side of the equitorial line. So, a cell with 46 molecuels of DNA (chromosomes) will produce two dauther nuclei (and then cells) with 46 molecules of DNA (chromosomes). They will be genetically identical.

Beyond showing the arrangment of chromosomes, the image above also shows the different types of microtubules: Kinetochore, Polar and Aster. Aster, meaning star (Greek), comes from the starlike appearance of these microtubules. This starlike arrangment is also seen in the plant genus Aster, which is noted for the radial startlike flower petals. Note: they are sometimes referred to as Astral microtubules (Astral is an English adjetive derived from Aster).

Once the chromsomes have been arranged, they can be seperated. Anaphase is when there is visible seperation of the chromosomes into daughter chromatids. This link to an Anaphase Image is an excellent reference for what occurs in Anaphase (remember that you have motors that can move down microtubules). The image to the image to the right is a good quick visual of what starts to happen: the seperatation of chromatid. NOTE: At this point, there is no more chromosome; all we have left are the daughter chromatids. It is not uncommon though for people to start referring to these chomatids as chromosomes. As this can lead to a great deal of confusion, I want you to remember that at the end of anaphase we have daugter chromatids, not chromosomes.


During Telophase, the cell returns to normal interphase operation. The kinetochore microtubules will be released and begin to dissociate. The nuclear envelope will reform as the nuclear lamella (intermediate filaments) begin to reassociate. Once the protection of the nuclear envelope is reestablished, the DNA will be released from the supercoiled packing that produced the chromatids and chromosomes. As this process continues, cytokinesis can begin.
Remember, cytokinesis, or the division of the cell, does not have to take place after mitosis. For the vast majority of cells, mitosis and cytokinesis are coupled processes, but not all eukaryotes undergo cytokinesis. In animal cells, the act of cytokinesis is directed by the cytoskeleton, which causes the membrane to be pulled toward the center (remember the location of those polar microtubules?). As the membrane is dynamic, when pulled together, the phospholipid bilayers will "snap" together, or fuse, resulting in a seperation of the membranes.

Plants and fungi, which posses cell walls, experience cytokinesis in a dramatically different way. Prior to division, the plant cell will have made numerous vesicles that contain the raw materials needed to create cell walls. These materials are held in an inert state, with inactive enzymes needed to form these materials into walls. As telophase begins, these vesicles begin to line up down the equator of the cell, and begin to fuse (what do you think triggers this action? will it trigger the enzymes?). As the vesicles fuse, cell walls begin to form. As more vesicles fuse (remember the membrane is dynamic), the wall continues to grow (cell plate). Eventually the membrane surrounded wall will fuse with the parental cell wall. Once the cell plate fuses with the parental wall, you have two new daughter cells.
NOTE: Plant cell walls are generally impervious to water flow. As a by-product of plant cytokinesis, and the development of the cell plate, you will find holes between the two new daughter cells. Known as plasmodesmata, these membrane lined holes allow for a continuous cytoplasm between cells. This allows for rapid movement of water and nutrients (including plant hormones) between cells (Symplastic Flow).

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Study Note

You will notice that above I gave you a number of links. Do you think that they are important for your overall understanding of this topic?

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Remember This: The following phrase will help you as we move through genetics. Remember it!

"Base complementarity is the foundation of all genetic processes."

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Daily Challenge

Describe the process of mitosis in your own words. Feel free to use images, just reference where you got the image. Remember that this leads to your milestone paper and exam, spend some time to build a personal description of mitosis.

Daily Newsletter: October 29, 2013 - Chromosomes

Daily Newsletter

October 29, 2013 Chromosomes

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Chromosomes

The word chromosome has a number of common means, but as biologists we need a strong (strict) definition of the word. For a biologist, a chromosome is a single molecule of DNA with associated packaging proteins, and is visible only during Metaphase and Anaphase of nuclear division. This second part is important; the chromosome is visible under light microscopy (the common form of microscopy). The chromosome is visible because it condenses (becomes tightly packed) during nuclear division (thus it is a eukaryotic phenomena).

We use the term chromosome to refer to a single molecule of DNA, but remember, in the strict sense, it only refers to a visible arrangement of DNA and proteins seen during nuclear division.

Another loose use is referring to the single, circular DNA molecule found in bacteria (or other prokaryotic DNA). As bacterial DNA is not held by the same packaging proteins, and has different packaging issues, the use of chromosome in this case is in error. The term genophore is a more appropriate term for prokaryotic DNA.

In eukaryotes, DNA must be packaged. One reason for this is to prevent physical strain or damage to the DNA molecule. The second reason is to conserve space. DNA is just too large when it is not packaged. The basic unit of DNA packaging is nucleosome. The nucleosome is a unit of packaged DNA, in which the DNA molecule is wrapped 1.67 times (~147 base pairs) around a core of 8 positively charged histone molecules (positive charge attracts negative phosphates).

These histone cores, when associated with Histone₁ can begin to supercoil into a fiber. A cascade of interactions causes more supercoiling which ultimately forms the chromosome.

The chromosome when it first forms during prophase of nuclear division is actually two freshly replicated molecules of DNA (we just copied DNA). The image below is a single chromosome composed of two sister chromatids:

Notice that the two sides of the Chromosome are individual DNA molecules that we refer to as Chromatids. Holding the two chromatids together is a protein rich region known as the centromere. At the centromere, we will find a molecular motor known as the kinetochore. We will see the operation of the kinetochore during cell division.

NOTE: You have some words here that sound familiar and often get confused. In your notes, make sure you have definitions of Chromosome and Chromatid. Work on using these terms correctly.

Here is a video to help you visualize the process: How DNA is Packaged

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Daily Challenge

In your own words, describe how DNA is packaged into a chromosome. Why is it important that we package DNA at the nucleosome and chromosome level?

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Other resources:

• DNA Packaging: Nucleosomes and Chromatin
• Comparison of DNA Structures

Daily Newsletter October 28, 2013 - The Cell Cycle

Daily Newsletter

October 28, 2013 - The Cell Cycle

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Reading:

The Eukaryotic Cell Cycle
This is an excellent overview of the cell cycle. This is considered supplemental to what is in your textbook.

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The cell cycle describes the life stages of a cell. It starts just after cytokinesis, and continues until the next cytokinetic (or mitotic) event. The period when a cell is growing between division events is known as interphase.

Interphase is divided into three distinct steps: G₁ Phase, S Phase, and G₂ Phase.

The G₁ Phase stands for Gap 1 Phase, and begins just after cytokinesis. Gap refers to no visible change in the cell. Another good term for G is growth, and that is usually what happens during this phase. The cell is building primary products, compounds such as amino acids, phospholipids, carbohydrates, triglycerides. These are describe as primary products as they are the cellular biochemicals needed to increase the cells size (biomass). Thus we generally consider the G₁ phase as the timethe cell increases in size. Another way to say this is that a major goal in this phase is the development of biomass.
[NOTE: In multicellular organisms, some cells will stop mitosis. They are locked in what is known as the G₀ phase. The G₀ phase is similar to the G₁ Phase, but the cell will never level leave this physiological stage. After reading this newletter, I want you to consider a question: Why would you stop in the G₀ Phase if you were never going to replicate again?]

When the cell gets a signal to divide, the physiology of the cell changes. Metabolic pathways for creating deoxyribonucleotides are unlocked (dATP, dTTP, dGTP, dCTP). The cell will use these deoxyribonucleotides to synthesize DNA (Replication). The production of enzymes needed to replicate DNA will also be begin. (Think back to cell signaling).

During the S Phase, cellular metabolism is focused on DNA replication (S stands for DNA Synthesis). This is a complex event, takes time, and has consequences if there is an error; so most of the cell's work is dealing with DNA replication.

Once S Phase has begun, the cell has committed its self to the process of Nuclear Division and Cytokinesis. You never replicate DNA without moving toward Nuclear division, DNA replication without nuclear division usually results in cell termination.

After DNA replication, the cell needs to build all of the proteins and compounds used during Nuclear Division and Cytokinesis. This is the goal of the G₂ Phase. The G₂ Phase will continue until the required components are constructed, and the cell receives the signal to continue. At this point the cell moves into the M phase (which stands for either mitosis or meiosis).

Regulation

The processes of the cell cycle is tightly regulated.
What happens in unregulated or uncontrolled cell growth?

The regulation is based on Signals (yes, we're back to signals). To maintain homeostasis, an organism must replace certain cells during its life time. For instance, humans replace skin and mucous membranes constantly. Hormones, such as insulin-like growth factors, are signals used to make sure the body maintains its self by replacing cells.

Inside of cells, there is an internal signal system based on the protein family Cyclin Depdendent Kinases (CDK). Note, this is a family of proteins involved in cell cycle regulation (they also have a few other functions). As the name implies, the protein needs a Cyclin to function. For example, CDK2 requires Cyclin E during G₁. (NOTE: Cyclinn is a family of protein signal molecules assoiated with the cell cycle).

CDKs are always produced in mitotically active cells. (What term do we use for a protein that is always produced?)
Cells don't always have Cyclin, but instead produce them in response to a signal. [Note: The signals differ depending on if you are dealing with multicellular, colonial or singal celled eukaryotes, so we are not going to get into specifics. As these control vital processes that could cause damage to cells, e.g., think cancer, they have a very complex signal transduction. Think of it this way, the cell has to have get numerous "permissions" before it starts producing Cyclin, so it is highly regulated. For the purpose of this class, we will just say that it is controlled by "Growth Factors".]

Throughout the cell cycle, there are time points referred to as Check Points. These check points are where regulation occurs. For example, to move from the G₁ Phase to the S phase, you need to produce a set of cyclins to induce the activity of CDKs. The Cyclin-CDK complexes can then phosphorylate proteins. Why would you need to phosphorylate proteins?

During the G₁ Phase, the check point determines if you turn on the production of deoxyribonucleotides and the production of replication complex enzymes. There are check points in the G₁ Phase, S Phase, G₂ Phase, and M Phase. Many of these check points are determinations of the health of the cell, or the correctness of DNA replication and chromosome condensation. The image to the right shows the major check points, as well as the cyclin needed to activate (Cyc D to make deoxyribonucleic acids and the replication complex, Cyce to start the S phase, Cyc A to make sure replication is occuring properly, Cyc B to make sure the cell is ready for mitosis). Note: the check point in mitosis (M phase) is not shown. The M phase check point is to ensure that chromosomes have properly condensed and migrated.
As you can tell, the check points are there to make sure that the process is occuring properly. Consider the Cyclin/CDK system as the Quality Assurance & Quality Control (QA/QC) officer of the cell.

G_o Phase (in case you missed it the first time around)
Some cells become non-mitotic at a given point in development. For example, nerve cells stop dividing, as do cardiac muscle cells. When a cell becomes non-mitotic, it shifts from the G₁ Phase to the G_o Phase. The cell can be locked from mitosis by blocking the genes for either CDK or Cyclin.

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Daily Challenge

In your own words, describe the cell cycle. Then answer the following question: How are CDKs related to cancer?

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Optional Reading

If your really into the regulation of the cell cycle, try out this article:
Cyclins and Cell Cycle Regulation

Special Edition: Newsletter October 25, 2013 - Scientific Thought in Biology

www.bologsu.us/BOLO

Special Edition Newsletter

October 25, 2013 - Scientific Thought in Biology

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 Scientific Thought

Each academic discipline has a specific way of viewing the world, a paradigm.  In the sciences, one of the major filters is the Hypetheticodeductive Model, a common example of which is the Scientific Method.  If we look at the word hypetheticodeductive, we can easily distinguish two critical concepts:  Hypothesis and Deduction.  These are the critical foundations of scientific thought.

A hypothesis is a tentative explanation of a phenomena that can lead to predictions that can be tested experimentally.  One trick to the hypothesis is that  it must admit to the possibility of being false (a hypothesis that has no way of yielding a false result is invalid).  For example, you read about a new herbal supplement that increases intellect.  Doing some web searches, you find more information from the producer.  You read in the fine print that "people who do not experience increased intellect in the first few weeks have toxins in their body that block the beneficial effects of this supplement."  They then give you a link to their herbal detox.  Was the statement about results a hypothesis?  Could you have a hypothesis that said some nebulous toxin prevents claims that there was a negative result?

The second word to consider is deductive.  Deductive reasoning starts with one or more premises.  These are general statements, such as a hypothesis, that are used to build a logical conclusion.  In science, part of the process is to gather evidence that supports the hypothesis.  The analysis of evidence (facts, data) allows us to build a logical conclusion that either supports or refutes the hypothesis.

This brings us to a few more critical concepts worthy of consideration.  Experimentation is critical to a scientist.  You could generate hypotheses all day, but you are not doing science if that is all you do.  In order to do science, you must put your hypotheses to the test.  Think of experimentation as a set of actions and observations performed in order to solve a particular problem or question.  In science, the context is to provide support or refutation of a hypothesis.

What does an experiment give us?  DATA (a synonym for data in this case is FACTS).  Data is used as evidence to support or refute the hypothesis.

Goal of Science

Essentially, the goal of science is to predict and control natural phenomena.  To do this, we use our senses (even senses enhanced by high-tech instruments).  Because of our reliance on sensory information, we are unable to describe the Truth of Reality, but we can create models of reality that allow us to predict and control natural phenomena.  For example, a doctor can look at your blood work, listen to your heart and lungs, feel your lymph nodes, and from all the tests, diagnose an ailment.  They can then prescribe a treatment to control or eliminate the ailment.

Scientific Thought in Biology

Biology is like the other sciences, our core model is the hypetheticodeductive model.  Unlike physics and chemistry though, we also rely on pure observation without a formal hypothesis as a critical pursuit to understand natural phenomena.

Consider a "Disease Detective" confronting an outbreak.  Do they go in with the idea of "let's first form a hypothesis as to the cause", or do they just go in, look at patients, collect patient vitals, as well as samples.  They then analyze the collected information and samples, and begin narrowing down what they have found.  From their experience and knowledge, they may quickly rule out causes, but this is still not a formal hypothesis.  They may have an informal hypothesis that it is a biological agent, but they are still very much working on pure observation.  Are they even experimenting at first?

Consider the observations of Charles Darwin during his time aboard the HMS Beagle.  Did he have a hypothesis he was testing, or was he just observing?

Does this mean that all we do is observation?  No.  A critical feature of modern biology is experimentation.  Understanding of genetics and molecular has led to the ability to test hypotheses we could only formulate 10 years ago.  Powerful computers have allowed statistical analysis of decades worth of observational data.  Experimentation and data analysis are still critical, but remember so to is observation.

Discussion

Dr. Ignaz Semmelweis is a figure rarely discussed in general biology, but his work as one of the founders of Infection Control is important.  His work also stands as an incredible example of the hypethetico-deductive model at work:
Read the following websites/articles.  It is suggested that you read them in order.

• Dr. Semmelweis’ Biography 
• Ignaz Semmelweis 
• Ignaz Semmelweis and the birth of infection control 
• Mortality rates at the Vienna General Hospital 

In the discussion forum for today, describe how Dr. Semmelweis' work with puerperal fever demonstrate the scientific method at work. Look at the hypotheses he generated, and consider whether these were strong or weak?  Does the data shown in the fourth link provide support to Dr. Semmelweis' final solution? Did Dr. Semmelweis have the correct cause, or were there other discovers that ultimately explain what was happening in the First Ward?  Discuss the concept of an Agent of Change; how did Dr. Semmelweis fail as an agent of change, and consider how you would stand as an agent of change.
NOTE:  Don't answer these as individual questions.  Consider and build a response.

Daily Newsletter: October 24, 2013 - PCR

Daily Newsletter

October 24, 2013 - PCR

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Polymerase Chain Reaction

In 1993, Kary Mullis won the Noble Prize in Chemistry for his work on the technology of Polymerase Chain Reaction (PCR).  By using the basic mechanism of replication, PCR allows scientists to amplify a few DNA fragments (or even a single fragment) into thousands of identical copies (you increase your number of identical copies 3-5 orders of magnitude).

PCR uses thermal cycles (regular changes in temperature that are repeated multiple times) to denature DNA, anneal primers, and replicate. Primers?  Remember that you must have primers for DNA polymerase to work.  In this case, we provide primers that are complementary to segments of DNA that we want to copy (we provide both a 5'-3' and 3'-5' primer so we can get both strands).  So the selection of primers is a critical feature to PCR work.

Notice, we are also using thermal cycles.  Can we use just any DNA polymerase?  No.  We have to use a heat stable polymerase.  For PCR, we use Taq Polymerase, originally from the thermophillic bacterium Thermus aquaticus.  The Taq polymerase from Thermus aquaticus is not the most accurate, so people have been using thermostable polymerases from other prokaryotes.  Question:  How can a prokaryotic polymerase be used to replicate eukaryotic DNA?  (Genetic Code is universal, and so are the molecules)

In the PCR process, DNA is denatured to single strands.  Primers are annealed (complementary base paring) to the DNA strands.  Taq polymerase then elongates until the cycle ends or your at the end of the template.  You then repeat the process again and again.

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Daily Challenge 

Describe how PCR is used in research, genetic engineering and forensics.

Daily Newsletter: October 23, 2013 - Telomerase & Electrophoresis

Daily Newsletter

October 23, 2013 - Telomerase & Electrophoresis

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Telomerase
The requirement for RNA primers creates a problem at the ends of the DNA molecule, specifically where a strand ends at the 3' (there is a 3' on each strand, remember the strands are antiparallel).  When you use this strand for replication, you must first create an RNA primer.

Recall, DNA polymerase has a major requirement.  It must have a free 3' end to build (it must have a free 3' -OH group).   Consider the drawing below:

The purple line represents the template strand from the parental DNA molecule.  We are looking at the 3' end.  When we replicate, it must be done in an antiparallel manner; we must synthesize 5'➝ 3'.  This leaves the 5' end as RNA.  After DNA synthesis is underway, the primer needs to be removed.  DNA polymerase δ does this by attaching to an Okazaki fragment and replacing the RNA primer (see image below).

DNA polymerase δ must have a 3' end to work from, so it uses an Okazaki fragment to start.  It lacks the ability to bind the 3' to a 5' end (it needs a Triphosphate), so we use ligase to connect the two fragments.

Consider again the end of linear DNA. 
There is no 3' end that DNA polymerase δ  can begin working to replace the RNA primer.  What happens to this end is that you just delete the primer.  So, the newly synthesized strand will be shorter.  This is a problem!  The image below provides a different perspective:

Ultimately, replication will result in degradation of the DNA if this is not fixed.  Eukaryotic cells have an enzyme known as Telomerase that can extend the 3' end of DNA with a repetitive sequence  of nucleotides.  The enzyme contains both RNA and proteins, and is considered a reverse transcriptase.  The RNA is a template for the DNA sequence repeat.  Vertebrates use the DNA sequence TTAGGG as the repeat, but you should note that the telomere repeat is different for different eukaryotes.  For example, most plants use TTTAGGG as the telomere repeat.  Here is an image of the process in action:

 

 



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Gel Electrophoresis
One of the most common analytic techniques for nucleic acids and proteins is Gel Electrophoresis.  Electrophoresis deals with the movement of particles through a fluid relative to a uniform electrical field.  Remember that all nucleic acids and proteins are charged (remember, they have the ability to hydrogen bond).  As a simple explanation, a uniform electric field will have a + side and a - side, so movement in electrophoresis is determined by the electrostatic profile of the molecules.

In gel electrophoresis, the fluid is a semi-solid matrix (i.e., a gel).  A very common gel is made from Agarose, which is one of the components of agar, which is used to make solid culture media (the other component is agaropectin).  The gel is placed in a buffer solution in a box that has the ability to create an electric field (you will have an anode and cathode).  Movement of nucleic acids and proteins will be from - to +.  The size and conformation of nucleic acid fragments and protein fragments will determine how far along the gel a fragment will move.  Larger fragments move slower.

Because agarose gel can vary in viscosity, a known standard (you know the size and conformation) are always used so that you can get an accurate measurement.  You compare the movement of your samples to the known samples in order to determine fragment size.  Below is an image showing the gel as it is being loaded with samples (samples go into wells), and how it as it is exposed to an electric field.

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Daily Challenge

Explain how an understanding of telomerase is important to cancer therapy and questions about the physiological aspects of aging?

Daily Newsletter: October 21, 2013 - DNA Replication

Daily Newsletter

October 21, 2013 - DNA Replication

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Textbooks have a tendency to make replication one of the most complext topics covered. With a tendency to throw all the current research and understanding at students, they rarely take a step back and try to explain it. This newsletters has two goals: 1) to help biology students understand DNA replication, and 2) to show you want is expected from a Biology Freshman/Sophmore.



Central Dogma of Biology

 Before we get into replication, let's take a step back and look at the three core genetic processes, a.k.a., the Central Dogma. The central dogma describes the flow of genetic information in a cell. The core idea is INFORMATION. You may recall some of our early discussion on DNA, and about base complimentarity and the directionality of the molecule. This will become important rather quickly, but for just this moment, I want you to concentrate on the fact that DNA carries information. Information on how to build RNA and Proteins, both of which will produce the phenotype (expression) of the cell. For this reason, DNA, RNA and Proteins are considered Informational Macromolecules. This means that the sequence of monomers contains infomation, e.g., instructions on how to build RNA and Proteins. Since it is critical, the sequence of nucleotides carries information.
As you can see from the diagram of central metabolism shown above, there are three processes: Replication, Transcription and Translation. Think about those words. They are words used in reference to languages and documents. When you replicate a docuement, you want to ensure that you are getting a faithful (or even exact) copy of the original.

When you transcribe, you are moving from one medium, e.g., spoken word, to another medium, e.g., text. If you watch news shows, they will tell you that transcripts of the show are available. Court reports make transcripts of the trial. You are taking the language from one medium (in our case DNA) to another medium (RNA). The language is still the same (i.e., nucleic acid lanugae), just in a different form. Does the transcript have to be 100% correct? You want it to be, but it is not as exacting as a replication.
Translation is where you change languages. Unless you're fluent in another language, you will need somoene to help translate, or at least a good translation dictionary. Now you are moving the context from one language (nucleic acid) to another language (amino acids).

All of these processes rely on one key feature of nucleic acids: BASE COMPLIMENTARITY. In DNA: A complements T, and G complements C. In RNA: U complements A, and G still complements C.

Replication

In replication our goal is to take one molecule of DNA and make two daughter molecules of DNA that are identical to the first. Even the best replication processes can produce errors, but our goal is to be error free. This takes precision! As DNA is long, we also need this to be a fast process.

The enzyme that is ultimately responsible for replication is DNA Polymerase. [NOTE: there are multiple types of DNA Polymerase, but for now you just need to understand the core concept common to the DNA Polymerase family.] DNA polymerase is only one component of the Replication Complex, which is a complex association of proteins needed to successfully complete the replication event. Your goal at this time is to concentrate on DNA polymerase; we will talk about some of the other components later.
DNA is a double stranded molecule, in which the strands are anti-parallel. This means that one strand starts at the 5' end → 3' end, while the the other strand is revered. This can be seen in the image to the right. A common way of saying this is that we read DNA in the 3' → 5' direction. Why is this important? DNA Polymerase can only read DNA in the 3' → 5' direction, and can only build a new strand in the 5' → 3' direction. Before we go on, let's look another time at how deoxyribonucleotides are polymerized.


To the left you will see a generic image showing DNA Polymerase adding nucleotides to a growing DNA strand. Look carefully: A deoxyribonucleotide triphosphate (dTTP) is being added. The 5' phosphate of the new nucleotide is what will be used to form the phosphodiester bond. DNA Polymerase requires a free 3' end on which to grow the new DNA strand. To the right is another image that will help you with this concept. At this point, the critical thing to remember is that DNA Polymerase will need a free 3' end on which to add a new nucleotide.
This requirement to build in only one direction (5' → 3') creates a problem for the DNA process: the two strands read in opposite directions, and each must be replicated. It was noticed that one strand appears to replicate continuously, while the opposite strand appears to replicate discontinuously.

The original strand that reads 5' → 3' can be used by DNA polymerase to continuously produce the new 5' → 3' strand (the antiparallel complement to the original strand). We refer to the continuous synthesis strand as the Leading Strand. The other original strand, which read 3' → 5' cannot be copied continuously. A section will have to be exposed, replicated, and then another section exposed. This strand is constructed discontinuously, and is reffered to as the Lagging Strand.The image above shows the leading and lagging strand. Notice that the leading strand is replicating toward the Replication Fork(where the original strands seperate). As more DNA unwinds and opens, DNA synthesis continues down the leading strand.
The lagging strand though has to build in a start-stop action, producing Okazaki fragments. These fragments have to be sealed (phosphodiester bonds) together before DNA can rewind into the α-helix.

In the image above, you will notice a number of enzymes on the lagging strand. These enzymes are required for the initiation of DNA polymerazation, and then sealing the fragments. Looking at the image, you will see an enzyme called Primase, and a structure in red known as a Primer. Another restriction on DNA polymerase is that it must have a free 3' end from which to start building. DNA Polymerase is prevented from building a DNA strand from nothing. Something (a primer) must be in place upon which DNA polymerase can build. The Primer is constructed from RNA, and is a temporary scaffold upon which DNA polymerase can start working.

Eventually the primer will need to be moved. This is where you need to learn a little more about DNA polymerase. There are multiple DNA polymerases in eukaryotic systems. The general work horse of replication is DNA Polymerase III (DNA pol III), which is used to make long strands of DNA. DNA Polymerase I (DNA pol I) is used to replace primers (it is also used in repair functions). Even after the RNA primer is replaced with DNA, there is still a gap between fragments. Ligase is the enzyme used to create a phosphodiester bond between fragments, thus sealing the new sugar-phosphate backbone of the synthesized strand.

The result of DNA replication is that one molecule one DNA was used to create two new molecules of DNA. The two strands of the original DNA molecule became the template from which to build new complimentary strands of DNA. This is referred to as semi-conservative replication, as each new molecule has one strand from the original molecule, and one freshly synthesized complimentary strand.

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Daily Challenge

In your own words describe the purpose and process of replication.

Daily Newsletter: September 21, 2012 - Signal Amplification

Daily Newsletter

September 21, 2012 - Signal Amplification

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Resource

Signal Amplification - An animated tutorial produced by McGraw-Hill Publishing. (click on the link)
Secondary Messangers - A section from Cell Molecular Biology by Lodish H, Berk A, Zipursky SL, et al. (2000) from the National Center for Biotechnology Information's Bookshelf. (click on link)

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Another important secondary messanger system is the IP3 (Inositol 1,4,5-triphosphate) pathway. A primary purpose of this pathway is to release Ca²⁺ that is stored in the Endoplasmic Reticulum. Ca²⁺ becomes an activator of various proteins. An interesting aspect of this system is that IP3 is derived from phosphatidylinositol 4,5-bisphosphate (PIP₂), a phospholipid found in cell membranes. The system then uses a membrane component as the signal agent. The system also requires a second lipid component of the membrane known as diacylglycerol (DAG). To the right is a general schematic of the system from Wikipedia Commons.
 
Phospholipase C is the enzyme that converts PIP₂ into IP₃. It is also generally the signal receptor. IP₃ is water soluble, and will bind to the IP₃ receptor on the Endoplasmic Reticulum, opening CA²⁺ channels. Calcium ions are then able to activate Protein Kinase C with the help of DAG. At this point, we have phosphorylation of substrates (mainly other proteins).
The signal increases the presence of IP₃ in the cell, which changes calcium ion concentrations in the cells. An increased [CA²⁺] provides a greater probability that Protein Kinase C will be activated, resulting in a physiological change to the cell.

Daily Newsletter: October 15, 2013 - Direct vs. Indirect Signals

Daily Newsletter

October 15, 2013 - Direct vs. Indirect Signals

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Signal transduction involves a signal contacting an appropriate receptor which ellicits a cellular effect. The transduction is thus the changing of signal into an effect. Transduction can be either direct or indirect.
A direct transduction event implies that the receptor ellicits an immediate effect on the cell. A good example of this is the Androgen (Testosterone) Receptor. The image below is a good image of the basic signal transduction pathway. Testosterone is lipid soluble, so can easily move through the membrane. Once inside of the cell, it is changed to dihydroxytestosterone (DHT). This binds to the receptor, and then the receptor-ligand complex is moved into the nucleus. Inside of the nucleus it directly affects the transcription of genes. In this case, the receptor-ligand complex has a direct effect on the cell.

Indirect signal transduction will involve the creation of a secondary messanger. The receptor-ligand complex will create a condition where an intracellular chemical signal is created. One primary signal binding to the receptor can then be amplified within the cell by creating this secondary messanger. Below is a general diagram showing the use of a G-Protein interface. The Ligand-Receptor complex directly activates the G-Protein. The activated complex then activates a specific effector in the cell membrane.

























In the above image, there are four views of the Receptor-G-Protein complex. In the top image of the cycle, the Resting State, there is no ligand bound to the receptor. When the ligand binds (the next image in the cycle), GTP binds to the Gα subunit. This causes the Gα subunit to detach from the rest of the G-protein (Gβγ Subunit). The subunits then bind to Effectors.
A very prevelant effector enzyme is Adenylate Cyclase. This enzyme takes ATP (Adenosine Triphosphate) and removes two phosphates. The remaining phosphate binds back to the ribose sugar at the 3' carbon, creating a ring structure (cycle) between carbon 5' and 3'. This is known as cyclic AMP (cAMP). cAMP is a very common secondary messanger. It can be produced in bulk and can bind to a variety of enzymes.

A good example of a cAMP regulated protein is Protein Kinase A (PKA).  This is a cAMP Dependent Protein Kinase.  The function of this family of proteins varies depending upon the cell (i.e., the current proteome of the cell).  For example, in adipose tissues, PKA activates pathways for lipolysis (breakdown of triglycerides), while in skeletal myofibers (myocyte or muscle cell) you see activation of glycogenolysis (breakdown of glycogen), inhibition of glycogenosis (making of glycogen), and increased rates of glycolysis.

Why would phosphorylation affect protein activiation? Phosphates are large with a -2 charge. Binding a phosphate to a protein will force the protein to change shape (configurational change).
Below is an example of indirect signal transduction via a G-protein:




Here is another picture of the G-protein to help you visualize the process.

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Daily Challenge

Using Testosterone and Epinephrine as examples, explain the difference between direct and indirect signal transduction

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Here are a few videos to help you better understand the processes discussed. 

Daily Newsletter: October 14, 2013 - Basics of Cell Communication

Daily Newsletter

October 14, 2013 - Basics of Cell Communication

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This week, we come to the end of building our mental picture of the fundamental unit of life: the cell.  What we have done up to now is look at the building blocks that make up cells: the phopholipid bilayer with all of the associated membrane proteins; how proteins are formed, and how they work; how cells acquire energy and carbon; and now, how they interact with their environment.

Cells must be able to sense their environment and respond to environmental stimuli.  The environment could be the intersitial fluid bathing the cells of your body, the moist soil around a plant root hair, or even the old cheese in your refrigerator that is now starting to mold.  Remember that all cells strive for homeostasis, and being able to respond to changes in the environment helps them to maintain and balance their dynamic metabolic equilibrium.

In order to maintain homeostasis, cell require some mechanism to receive environmental signals, and then process those signals into a response. All cells, prokaryotes and eukaryotes, have this ability. Beyond just responding to the environment, we now know that cells, even bacteria and archaea, possess the ability to signal each other. In multicellular organisms, we will talk about the coordination of metabolism and growth using chemical signals. In humans, from the embryo stage til death, cells are constantly talking to each other.

Signal reception occurs in all known organisms (from picking up environmental to cellular signals). For instance, in bacteria, we know of a signal phenomena known as Quorum Sensing. With this signal system, bacteria release chemicals as they grow which other individuals of the same species (and some times other species) pick up; when the concentration of the chemical reaches a critical point, cellular changes can be observed in the community. Basically, as the population increases, cells begin to change.

But how do cells pick up signals? Signal recognition begins with receptors (generally protein in nature; many are glycoproteins).

For a cell to pick up, or register, a signal, it must build a receptor for that signal.
(IMPORTANT NOTE: a cell that lacks a receptor for signal X can not register signal X; they are deaf to the signal). This sets up another question: what is a signal?

Most of the signals we will talk about are chemical signals (aka, Ligands), meaning we have a chemical compound that will "fit" a receptor, activating it. There are other signals though: light can be a signal (photoreception), temperature (thermoreception), and even mechanical such as touch (mechanoreception). As mentioned, out discussions for this week will focus on chemical signal pathways.
By far, the most common type of signal system will involve chemicals. Hormones are chemical signals, neurotransmitters are chemical signals, even carbon dioxide is used as chemical signal in the human body. Because there are so many different chemical signals, we have a generic word for any compound that could bind and activate a receptor protein: Ligand. As a general word, ligand is used when we discuss the basic concepts of signal systems. (NOTE: In biology we have a number of GENERIC words that are used in discussing basic pathways or models. Ligand is one of those terms).
At their most basic, a chemical signal system (or pathway) will be comprised of a Ligand and a Receptor. When a ligand binds to a receptor (ligand-receptor complex), the receptor changes shape (conformation), which elicits a physiological response in the cell. Remember: The receptor is based on a  protein, and when proteins change shape, they have an effect on the cell. So the basic signal system will be: Ligand binds to receptor, receptor changes shape, cellular response occurs. This is known as Signal Transduction.

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Ligands are chemical signals, and receptors are based on proteins. The receptor is folded so that it forms a binding space (active or binding site*) where the ligand can dock and bind. A common thought is that every receptor has a specific ligand, and that nothing else binds to the receptor. A lovely fiction.

To the right is an image of the μ-Opioid receptor (μ is the Greek Letter Mu). This receptor is found in human neural tissue, and provides analgesic effects and feelings of euphoria. It can also cause respiratory depression and reduced GI motility. The main ligands for this receptor are enkephalins and β-endorphin. Yes, this receptor set has two potential ligands. It also has a list of agonistic and antagonistic compounds that can bind to the receptor.

When we speak of receptors and ligands, we talk about the affinity of the receptor for a particular ligand. Remember, we have folded the protein to create a 3-D shape. In the case of the μ-Opioid receptor, the folded protein has a notch where the ligand can slip in. This notch will have chemical properties complementary to the ligand.

A high affinity would imply that the physical shape and chemical properties of the notch are a good match to a given ligand. In this case, beta-endorphin is a strong chemical and physical match to the binding site (notch) of the μ-Opioid receptor. Dynorphins, which are another type of neurotransmitter (ligand) in the brain, have a low affinity for the μ-Opioid receptor's binding site, meaning the shape and chemical properties are not a good match.

This sets up another aspect of receptors: agonists and antagonists. These terms represent chemical mimics of the natural ligand(s) of a given receptor. Agonists are chemical mimics that bind to a receptor and trigger a cellular response; in other words they work like the ligand. Antagonists on the other hand are chemical mimics that bind to a receptor and block a cellular response. In fact, antagonists can stay bound and prevent activation of the receptor. A well known agonist for μ-Opioid receptor is morphine. Morphine can bind to the μ-Opioid receptor and active the cellular response that leads to analgesic effects, feelings of euphoria and respiratory depression. To the left is an image depicting agonistic and antagonistic bindings possible with the μ-Opioid receptor. NOTE: The agonist relationship implies an activation of the cellular response, while the antagonistic relationship implies an inactivation of the cellular response.

Morphine is a powerful analgesic with many well known side effects. One of the most dangerous of these side effects is physical addiction and an increased tolerance for the drug (you need more and more to get the same effect over time). This provides a good example of another principle of receptors: Regulation.

Down Regulation: When a cell receives too much signal, it will begin to down regulate the receptors. This means that the cell stops producing the quantity of the specific receptor it usually makes. For membrane bound receptors, over time, as the membrane is repaired and refurbished (a constant dynamic process), the number of expressed receptors drops. The result is that the cell is less sensitive to the signal. Why does a cell do this? Think of it as a person being exposed to loud noises. If it happens once, for a short period of time, the body can compensate. What if the person is continuously exposed to the loud noise? Eventually they become less sensitive to sound, i.e., they become functionally deaf. The same thing is happening to a cell that is overexposed to a ligand. To protect themselves, they produce less and less of the given receptor. In some cases this becomes an irreversible loss of the receptor from the cell. In the case of the μ-Opioid receptor, your body produces only small temporary doses of β-endorphin. With morphine, you have a large dose, and it is usually for a long duration. The longer the duration (over a week), the more likely you will have desensitization (down regulation) of the receptors.

Up Regulation: The reverse of down regulation is up regulation. If a cell is not getting enough signal, it will start building more receptors. In this case, there is a deficency in the amount of the ligand preset. The cell is compensating by building more receptors.

Both Down Regulation and Up Regulation are examples of Negative Feedback.
*NOTE: Receptors and Enzymes are both proteins. They both have a site where a ligand (receptor) or substrate (enzyme) can bind. As with receptors, we will see that enzymes have an affinity for their substrate, and like receptors, other chemicals can bind into the enzyme. In addition, like ligand-receptor, when a substrate binds into an enzyme, the enzyme will change shape.

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Words of the Day: Paracrine & Autocrine

Prepare definitions for these two words and put them in your notes.

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Daily Challenge

Below is a diagram of the Insulin Receptor and Signal Transduction. Review the image and information in your text, then write a forum post describing the nature of the Ligand and Receptor, and how a signal can change the physiology (active metabolic pathways) of a cell.