So Darwin had A Big Idea ™ which has caused a ridiculous amount of debate/screaming/studying over the past 150 years. Everyone knows that, right? It’s caused so much uproar that recently the Texas school board has had near never-ending debates about the merits of teaching such Big Ideas to poor innocent children.
(Did you notice that I called it an “abstract?” You see, Darwin had been working on his book for over 20 years when it finally came out–and it only came out when it did because another young scientist had happened upon the same idea and Darwin didn’t want 20 years of thought and work to go down the drain. So instead of publishing the 1200 page opus he originally planned, Darwin cut it down to a svelt few hundred pages and called it an abstract. Just an historical tidbit).
Anyhow, Darwin’s ideas have not been taken at face value since the moment his pen hit paper. As with most scientific ideas, they have been analyzed, discussed, tested, reworked, and improved upon until they are almost unrecognizable in their original form. This is why I love science so much–the ideas are constantly changing and improving, and each member of the community has the opportunity, nay, duty, to suport or falsify the concepts that came before.
But that’s a bit too “philosophy of science” for this post. No, what I’m attempting to explain here is the concept of modern evolutionary synthesis, commonly called “the new synthesis” or “neo-Darwinism.”
So, in 1859 Darwin published On the Origin of Species, and it sparked a huge reaction. His main ideas can be summed up in four major points:
1. There is variation within species
2. These variations can be passed on to offpsring
3. Each generation sees too many offspring born than can possibly survive
4. The survival and subsequent reproduction of offspring isn’t random; those that do survive long enough to reproduce, or those that reproduce the most have the most favorable characteristics. Those characteristics are passed on to offspring. Those characteristics are naturally selected.
What’s interesting is Darwin did not put for the idea of evolution–in fact, the word “evolved” is only used once in the entire book, and is the last word of the last chapter. Evolution as a concept had been around since the mid 1700’s, and was generally accepted even by the devoutly religious as a plausible explanation of animal life. What people didn’t know was HOW evolution worked, and there were many scientists putting forth various hypothesis to explain the mechanism.
Darwin came up with his ideas after years on board a ship, and collecting and observing all manner of life forms across the world. He then let the idea fester in his mind for over 20 years and slowly developed the over arching idea that evolution occurs through small variation from generation to generation, and that the most beneficial variations are conserved through natural selection.
The biggest problem with the idea of natural selection back in Darwin’s day, however, was no one could quite figure out how it actually worked. The evidence made sense (and Darwin does a beautiful job of beating the reader over the head with chapter after chapter of evidence for natural selection), but the actual mechanisms of inheritance still eluded the best scientists of the time. The debate raged on.
Meanwhile, off in Germany, a little known monk by the name of Gregor Mendel was doing some experiments with pea plants.
Mendel figured out that traits were passed from generation to generation in a mathematically predictable manner. The problem was few people knew about his work. He published it in 1865, about 6 years after the first edition of On the Origin of Species, but all evidence suggests that Darwin never read the paper. Both Darwin and Mendel died before someone figured out to link the two ideas together.
But get linked together they did (Yay for literature searches!!). The 20th century saw some clever person pick up both pieces of work and think “Huh. This seems to work well together!” Mix in the discovery of genes and the idea of population genetics, and you get an explosion of evolutionary ideas that united a formerly divided group of scientists.
After a few decades of experiments and nobel prizes, the New Synthesis was born, and incorporated the ideas of both natural selection and genetics:
1. Populations contain randomly derived genetic variation accomplished through mutation and recombination
2. Gene frequency within populations changes through genetic drift (allele frequency change due to random sampling), gene flow (the transfer of alleles from one population to another), and natural selection
3. Most adaptive genetic variants have slight phenotypic effects, so change in phenotype is gradual
4. Speciation involves gradual evolution of reproductive isolation among populations, and is what causes diversification
5. The processes of speciation over a long enough time cause changes so great that higher taxonomic levels are necessary (genera, families, orders, etc.).
Basically, the modern evolutionary synthesis is the application of Darwin’s idea of natural selection to Mendel’s idea of inherited characteristics, with a little bit of Watson and Crick mixed in.
Of course, listing the 5 major ideas of any paradigm simplifies it a little too much. These ideas were created by numerous scientists over the course of a decade, and represent some of the greatest ideas evolutionary biologists have had.
The major names associated with modern evolutionary synthesis are Julian Huxley, R.A. Fisher, J.B.S. Haldane, Sergei Chetverikov, Theodosius Dobzhansky, E.B. Ford, Ernst Mayr, Sewall Wright, George Gaylord Simpson, G. Ledyard Stebbins, and Bernhard Rensch.
Julian Huxley is credited with coining the term “modern evolutionary synthesis” in a book he wrote in 1942 Evolution: The Modern Synthesis. Julian Huxley was the grandson of Thomas Henry Huxley, the man known as “Darwin’s bulldog” due to his dogged defense of Darwin’s ideas. So I suppose it’s no surprise that Julian Huxley would carry on that tradition two generations later by bringing Darwin’s ideas into the 20th century. Julian’s book merges the revolutionary ideas of population genetics and genetic inheritance with natural selection by approaching genes from a natural selection standpoint. The book has two major editions, and has just recently been rereleased.
Of course, Huxley did quite a bit more than simply write a book–the years up to the writing of this work were filled with evolutionary research and the years after were filled with the same. He will forever be known as the man who coined the term, however.
R.A. Fisher is the guy responsible for such statistical break throughs as Fisher’s exact test and the ANOVA. I blame him for making stats such a pain in the butt! And so very useful. Thanks Fisher! Anyhow, Fisher was an active mathematician and geneticist during the first half of the 20th century. His interest in eugenics, mathematics, and evolution gave him the tools to become the founder of quantitative genetics. He spent much of his time attempting to calculate the distribution of gene frequencies among populations. Geez! I also had problems with those calculations in my molecular ecology courses! Apparently this guy is responsible for all the difficult homework I’ve had over the last two years. Thanks, Fisher.
JBS Haldane is the second of three important mathematicians that helped to quantify rates of changes in gene frequencies (the first being RH Fisher). In 1932 Haldane penned a book entitled The Causes of Evolution, which summarized his work on the mathematical theory of natural selection. Haldane went on to write an essay called On Being the Right Size, which postulates that physical size is often what determines the equipment necessary for life in a given species. This idea is referred to as Haldane’s principle by modern biologists. On a humorous note, Haldane famously answered the question “What can be inferred about the mind of the Creator form the works of His Creation” with “An inordinate fondness of beetles.”
Sergei Chetverikov was one of several Russian scientists that ventured into the world of evolutionary biology in the early 20th century. Chetverikov worked with fruit flies, and in 1926 wrote a paper that bridged early theories of genetic evolution with real world populations. He found that the process of mutation was the same in natural, laboratory, and domesticated populations; most mutations are deleterious but there ere some that do not reduce viability; a randomly mating population is stable; new mutations are absorbed by heterozygous individuals; mutations gradually spread through the population by chance (a very early concept of genetic drift); and isolation and genotypic variability lead to differentiation. These concepts are just a few put forth in his major work, which, unfortunately, was only published in Russian. Luckily, Haldane had a translated copy and was able to make use of these concepts during the new synthesis.
Theodosius Dobzhansky is another influential Russian scientist, although he had the distinction of moving to America during his seminal research years. Dobzhansky, a geneticist by trade, is credited with writing a major work on modern evolutionary synthesis: Genetics and the Origin of Species. This work defines evolution as “a change in the frequency of an allele within a gene pool” and is responsible for spreading the idea that natural selection takes place through mutations on genes. He is also credited with the famous phrase “Nothing in Biology Makes Sense Except in the Light of Evolution,” which is the title to a famous article in which he articulates the conflict of evolution and creation.
E.B. Ford was a student of Julian Huxley at Oxford, and spent his career working on insects. He is responsible for the field of ecological genetics, and was on good working terms with Dobzhansky and Fisher, with whom he exchanged numerous letters and ideas. Ford formalized the definition of genetic polymorphism (when two or more clearly different genotypes exist within a single species), and used his knowledge of Lepidoptera to test and eventually prove many of Fisher’s predictions. Ford’s most famous student was Bernard Kettlewell, who conducted experiments on the evolution of melanism in the peppered moth, an experiment which delights and annoys students to this day. On a side note, Ford campaigned for the legalization of male homosexuality in his native Britain. Just an FYI.
Ernst Mayr (pronounced “Mire”) was a taxonomist and naturalist who was the youngest of the modern synthesis boys (he died only recently in 2005). His major book was Systematics and the Origin of Species in 1942, in which he helped to define the biological species concept: a species is not just a group of morphologically similar individuals, but a group that can breed exclusively among themselves. He suggest the concept of peripatric speciation, in which populations of adjacent yet isolated individuals will, though genetic drift and natural selection, evolve into distinct species over a period of time. Mayr was a voice of dissension among the modern synthesis crew, insisting that natural selection acted upon the whole organism and not individual genes. He also criticized molecular evolutionary studies. Huh. Maybe he was kind of a jerk. A smart jerk, but a jerk none the less.
Sewall Wright is the third mathematician that I blame for all those damn calculations I had to do in molecular ecology. He worked with Fisher and Haldane on the concept of population genetics, and discovered the inbreeding coefficient.
George Gaylord Simpson (hee! Gaylord) was an influential paleontologist during the early 20th century, and lent a much needed historical perspective to the new synthesis. He wrote several works including The Meaning of Evolution and The Major Features of Evolution, and was an expert on extinct mammals and their migratory patterns. He is known for coining the term hypodigm (a sample from which the characteristics of a population may be inferred), and he predicted the concept of punctuated equilibrium years before it was officially put forth by Dawkins.
G. Ledyard Stebbins is the lone botanist in this crew, and studied the evolutionary biology of plants at Berkeley. He wrote Variation and Evolution in Plants, which, by many, is considered a core of the evolutionary synthesis. His work on polyploidy and speciation in plants has influenced nearly all botanists since his time. When speaking of his own work, he never considered his contributions unique; he simply thought he was applying evolutionary biology and genetics to plants, and describing how these concepts affected botany as a whole.
Bernhard Rensch is the lone German of this group, and is responsible for popularizing the new synthesis in his native country. His primary work involved explaining how the concepts that drove speciation could be used to describe higher taxa. He also worked in areas of animal behavior.
It’s almost magical how just the right people with the right backgrounds in the right areas all were studying at the same time, isn’t it? It’s a tribute to the beauty of the scientific method, I think, to see how each man’s ideas were disseminated and used by the others to eventually create a paradigm shift in the way biologists think about evolution.
So I’ve taken this week to focus on information required by my microbiology professor :”Know about biofilms and quorum sensing.” No detail on *what* I should know about those two huge topics, but I need to know about them. All of it, I guess.
I guess I’ll work on just knowing what I can. Let’s get to it, shall we?
First up, my plan of attack. There’s a lot of information out there about biofilms (they’re the darling of the microbiology world at the moment, and lots of time and effort is going into studying them), so there’s no way I’ll be able to memorize everything about them in the next few weeks. What I can do, however, is focus on a model specimen, and know everything about that particular model.
Pseudomonas aeruginosa is a bacterium that is frequently found in biofilms, and happens to be one of the most studied species in the biofilm world. (This is probably due to its annoying tendency to infect humans).
This makes it the perfect subject for me to use as my model specimen. Let’s get to it!
First and foremost: what are biofilms?
Biofilms are communities of microorganisms embedded and immobilized in an extracellular polymeric substance (EPS) attached to a solid surface. The biofilm gives microorganisms protection from things like desiccation, predation, and antibiotics, and allows them to share water and nutrients. It has also been proposed that biofilms allow for the sharing of DNA, thereby facilitating the transfer of beneficial genes from cell to cell. This community organization is in direct opposition to the way we have studied microorganisms for many years: as free living single-celled organisms (called planktonic organisms).
Biofilms are highly variable, and their exact make up and structure changes with species, temperature, stress, nutrients, and other environmental factors. While this is super annoying for those of us who want to know everything about biofilms, it’s actually a very good strategy for the micro communities that utilize these biofilms as environments: great diversity keeps a single organsim from decimating the world-wide biofilm population.
What makes a biofilm a biofilm is the presence of the extracellular polymeric substance (EPS for short).
This stuff was originally called “extracellular polysaccharides” until scientists figured out that there were lipids, proteins and DNA involved as well. Luckily, they were able to come up with a phrase that kept the EPS acronym, so everything worked out in the end.
In order to form biofilms, planktonic bacteria go through five primary stages of biofilm development:
1. Initial attachment
2. Irreversible attachment
3. Maturation I
4. Maturation II
I’ll look at these in order, using P. aeruginosa as a model organism.
1. Initial attachment.
First and foremost, the planktonic bacteria need to get to the surface of something in order to form a biofilm (remember that the definition of a biofilm is a community of bacteria embedded in an external matrix attached to a solid surface). Pseudomonas aeruginosa does this via diffusive transport
and active transport driven by bacterium flagella.
Once the bacterial cells arrive at an appropriate spot, they attach to the surface. They are able to attach due to their flagellum, type IV pili, extracellular DNA, and Psl polysaccharides.
The flagellum is involved in attachment due to its stators.
You see, the flagellum works by spinning. There’s a spinning rotor portion, and a stationary stator portion, within which the rotor spins. In P. aeruginosa there are two stators, and both must be present for biofilm attachment. Truthfully, though, we don’t know exactly what the stators do to facilitate this attachment–we just know that mutants that don’t have the stators (either one or both) are unable to attach to surfaces and form biofilms.
Extra cellular DNA (eDNA) is an interesting discovery–scientists knew that it existed, but thought it was simply an artifact of lysed cells in the EPS. However, in depth studies of P. aeruginosa revealed that biofilm formation was impossible without it, and the bacterium produced such large amounts of eDNA by creating releasing vesicles from the cells which house DNA.
So far, eDNA is simply refereed to as an “adhesion factor.” This is science code for “we know it’s important, but we don’t know how or why.” Basically, scientists have found that when cells don’t make eDNA, or when bacteria are flooded with something that breaks down the eDNA, these cells cannot form biofilms. If the eDNA is present, biofilms are present. Pretty convincing argument that eDNA is required, but there’s little information as to what it does exactly.
One article mentioned that eDNA may react with N-acyl-glucosamine, a major component of cell walls in bacteria. This reaction would cause a a bond to form between the eDNA and the cell walls, which would cause the cells to stick together. This may be the mechanism by which eDNA causes biofilm formation–flagella and type IV pilli are used to attach to a solid surface, while eDNA is used for cell to cell attachment. I wonder then if biofilms that are not attached to a solid surface (although some purists don’t consider these flocs biofilms at all) have a disproportionately high amount of eDNA? This is what’s neat about science…there’s so much to find out!
During this stage of initial attachment, some cells attach to the substrate and some don’t.
2. Irreversible attachment
This stage is characterized by most cells being attached, and the community differentiating into two parts: parts of the new community that are motile, and parts that are immotile. This seems to be a function of the nutrient availability of the environment where the bacteria are forming a biofilm. If there’s plenty of glucose in the environment (which P. aeruginosa uses as a carbon source), then part of the bacterial cells become immediately nonmotile, while another part are motile.
At this point, the Las quorum sensing system became active (that is the subject of the next blog, so I’ll let you wait for an explanation).
The nonmotile cells form microcolonies, which are used as “stalks” for mushroom-shaped structures. The motile bacteria climb up the stalks and form the “caps.”
If citrate is used as a carbon source, however, the cells are all motile to begin with, and none of them stop to form these stalks. The resulting biofilm is flat.
Man, phenotypic variation due to differences in nutrient availability! How much more complex can we get?!?
During this time, the extracellular polymeric substance is produced. The EPS is involved in attachment (naturally), interactions between subpopulations and therefore cell-t0-cell intercommunications, tolerance, and exchange of genes. As I said above, the matrix is made of polysaccharides, lipids, proteins and eDNA. We know the most about the polysaccharides, proteins and eDNA (it only has recently be discovered that lipids are more prevalent than we thought…that’s always how it goes, isn’t it?). The exact make up of this matrix is, once again, dependent upon environmental conditions. That makes sense, doesn’t it? The armor you wear, the communication you use, and the way you attach to things is always going to be dependent upon what is going on around you.
These different environmental conditions are sensed and regulated by a series of kinase/response regulators called LadS, RetS, and GacS.
Oh dear, new terms. Ok, what the heck are kinase/response regulators?
Let’s start with a kinase. Notice that -ase suffix on the word? That means it’s an enzyme. This particular enzyme is responsible for transferring phosphate groups from high-energy donor molecules (such as ATP) to some sort of substrate.
A phosphate group is just a phosphorus molecule with some oxygen and hydrogen attached.
When a phosphate group is added to another molecule or substrate, that substrate changes (duh…it’s got something added to it!). That change can change the shape of the molecule, opening up some receptor site that was closed before. Or it can allow the molecule to react with a new substrate. Whatever happens, the phosphorilation (addition of a phosphate group) causes a change in the substrate which starts a cascade of reactions within a cell.
It’s a neat system, actually…since cells don’t want reactions happening all the time, they can regulate them by requiring a phosphate group inorder to make a substance reactive. That phosphate group won’t be added with out kinase, and that kinase won’t even be around until the right time. Regulation at it’s finest!
Ok, now we know what kinase is, what about response regulators?
Response regulators are exactly what they sound like–proteins that regulate a response, usually the transcription/translation of a gene. They work with kinases by accepting the phosphate groups.
HDS=histadine kinsase; RR=response regulator; P=phosphate group
So, a kinase is usually in contact with the outside of the cell somehow. When it gets the signal it’s looking for, it takes a phosphate group and attaches it to a response regulator. The response regulator changes shape and becomes active, when then causes something in the cell to happen.
The kinase/response regulators involved in matrix production in biofilms are LadS, RetS and GacS. These three things are membrane associated proteins (proteins that span the membrane) and therefore search the environment for signals.
See those red things? Those are proteins embedded in the membrane.
What do the proteins search for? We don’t know. But it’s something, and when they find that special something it triggers the histadine kinase to phosphorelate the response regulators. In turn, the response regulators turn on groups of genes which do all manner of things, including producing the matrix.
Look at it go!
Once the matrix is in place, the cells are immobile and go on living as a community.
This formation of stalks and caps is characteristic of
3. Maturation stage 1
At this point, the layers of cells become greater than 10 micrometers thick.
The formation of mushroom caps involves the presence of some of the same things as initial attachment: eDNA, flagella, and type IV pili. The type IV pili may bind to the eDNA, while the flagellum allows the cell to get to its required place on the stalk.
During this stage, a second quorum-sensing system is switched on: the Rhl system.
This differentiation continues on until the thickness of the biofilm reaches a maximum of 100 micrometers, which introduces:
4. Maturation 2
This stage is characterized by the cell layers at their thickest points, the cells are nonmotile, and most of the cells are differentiated. At this point, the biofilm is at its most mature, and involves water channels, a highly organized system of cell colonies, and the ability to share genes among individual cells.
After several days of maturation, cells at the center of many of these clusters become motile and begin to disperse into the surrounding liquid.
Of course, the problem with attempting to characterize stages of development is that a mature biofilm may exhibit all 5 stages of development at any given time.
Well, that’s it in a nutshell. Biofilms are becoming more and more important in all manner of things, from medicine to engineering. I’m betting that this is the direction that microbiology will go in the future (and is probably why I need to know everything I can about the glory that is biofilm).
So for the past few months I’ve been frantically studying for not the GRE, but for my preliminary exams for my PhD. I will therefore be adding blogs that answer potential questions from my prelims in addition to GRE questions. This should be fun for all!
My advisors have been giving me “hints” (i.e. “know everything in the world”) about the type of questions they are going to ask me, and one such hint made it perfectly clear that I better have a very clear understanding about the techniques I am using in my research. I won’t go into detail, but my research involves flies and bacteria, and I’m identifying that bacteria with pyrosequencing. I’ll then figure out the concentration of bacteria using qPCR or rtPCR, whichever our lab can afford. What the best way to understand these techniques? Explain them to you, good readers! Here we go.
First, a little bit of background. Both PCR and pyrosequencing involve DNA, and therefore you have to know a little bit about how DNA replicates. Long story short: DNA is made of long chains of 4 nucleotides: A, T, G, and C. The sequence of these nucleotides (called bases) determines which amino acids are produced, which in turn determines which proteins are produced, which then make up all that we see as life. Neat! DNA is packaged into cells in double strands…each strand is complimentary to the other. When these complimentary strands unzip, they can pair up with free nucleotides and make copies of themselves. This is what all these techniques are based on.
Ok, lets get into a little bit more detail about how this replication works exactly, shall we? Nucleotides are made up of a nucleobase (adenine, guanine, cytosine, or thymine-A,G,C or T), a five carbon sugar, and some phosphate groups. Don’t let those terms “5 carbon sugar” and “phosphate group” scare you–a 5 carbon sugar is just what it sounds like…a sugar with 5 carbon molecules arranged in a ring:
Hello there 5 carbons! Ok, we’ve got one of our bases attached to this ring of carbons, plus a phosphate group:
See? Phosphate groups are simple. I always get a little freaked out when scientists start changing the endings of words–all those “-ates” and “-ites” throw me off. However, they are really just word parts that tell me a bit about the compound. I won’t go into them here, but you can read about them on Wikipedia. Thanks Wiki!
Ok, so now we’ve got our complex compound: A,T,C, or G attached to that 5 carbon ring, which has some phosphate groups hanging on it.
Now, phosphate groups are reactive–they like attaching to what we call hydroxyl groups. Hydroxyl groups are simple things with confusing names (like most things in chemistry, I think). It is simply an oxygen bonded to a hydrogen. How simple is that?
So phosphate groups and hydroxyl groups totally love each other, and they want to bond ALL THE TIME. It’s actually kinda cute. And a little gross. Anyhow, nucleotides have all these groups in particular places on their carbon rings. Let’s look at that picture of the five carbon ring again:
Ok, see the numbers? We pronounce those numbers as “five prime” or “three prime.” On nucleotides, the phosphate group is attached to the five prime carbon. See it? A hydroxyl group is attached to the three prime carbon. When two nucleotides are lined up next to each other, the 5′ (five prime) phosphate group bonds with the 3′ (three prime) hydroxyl group, and they totally make out. And make long chains of nucleotides, which become DNA. Whatever.
Therefore, in order for a nucleotide to attach to the end of a chain of nucleotides, the 3′ end has to be exposed at the end of the chain. Don’t worry, you’ll understand why I’ve explained all this in a second.
Now that we have the basics of how DNA replicates and how nucleotides stick together to facilitate that replication, let’s move on to some of the procedures I promised I’d explain forever ago.
PCR is short for Polymerase Chain Reaction, and is a method we can use to clone sequences of DNA. We often want to clone these sequences a whole bunch (on the order of a billion copies of a single sequence!), so technology is obviously involved. This is how it works:
DNA is collected from somewhere. It can be anything, really, and we only technically need a single copy (although more DNA makes this much easier). We take that DNA and break it apart. Knowing what we know about the structure of DNA makes this process simple. The two complimentary strands of DNA are attached via hydrogen bonds. Heat can break those hydrogen bonds (this is one of the reasons living things can only tolerate so much heat–DNA actually breaks apart). The temperature at which the two strands of DNA disassociate is called the DNA’s melting temperature, and varies with the DNA sequences.
You see, base pairs are attached by slightly different bonds: A attaches to T via a double bond, while G attaches to C via a triple bond. The more bonds present, the more heat it takes to break those bonds. Therefore, GC bonds take more heat than AT bonds.
If a strand of DNA has a bunch of GC pairs, then it’s going to take more heat to cause the complimentary strands to disassociate. More heat means a higher melting temperature. But I digress.
The DNA is heated until all the hydrogen bonds are broken, and then we can focus in on the particular part of the DNA that we want to copy (called “amplify”). I suppose we could do the entire genome, but that would take FOREVER and use up a lot of reagents. We don’t want that. Let’s focus on a single gene, or section, or tiny little part instead, shall we?
So, what part do we amplify? Well, that depends on the question you’re asking. Most of the time you do PCR so you can identify a particular species, or look to see if two people are related, or identify a person, or something like that, and the regions you choose to amplify vary for each of these questions. My research involves using PCR to identify a species, so I’m going to look at a particular section of RNA called the 16S rRNA region. Let me explain (because you know you want me to).
All organisms, be them eukaryotic (having membrane bound organelles) or prokaryotic (no membrane bound organelles) have ribosomes. Ribosomes are those parts of a cell that take amino acids and knit them together into proteins. Without ribosomes, proteins would never be made, and life as we know it wouldn’t exist. Thanks ribosomes!
Anyhow, ribosomes are able to do what they do because they are made up of RNA (RNA is that compliment to DNA that takes information all over the place). The RNA in ribosomes is broken up into two subunits: a large subunit and a small subunit, with messenger RNA (mRNA) smashed between the two subunits.
The amount of RNA in the ribosome depends on if the organism is a prokaryote or a eukaryote. Eukaryotes have larger chunks of RNA, and therefore the subunits are larger. The size of RNA is measured a little strangely–it doesn’t have to do with length or weight or mass or some simple measuring tool like that. Things this small are hard to measure with a ruler anyhow. No, RNA is measured by where it floats in a liquid while spinning in a centrifuge: the bigger it is, the lower it will sink when spun around. The smaller it is, the higher it will float.
Think of it this way: you know those spinning roller coasters at amusement parks where you stand against a wall and then the floor drops out and you stick? The ones where they say “if you are going to vomit, cover your mouth and raise your hand!” because if someone pukes EVERYONE is gonna have a bad day?
You ever been on one of these? They’re super fun. Did you ever look around while it was spinning, though? If you had, you would have noticed that larger people tended to slide down the wall (sometimes coming to rest on the floor ), while smaller people could stay really high up on the wall. This is a good way to separate the really big people from the really small people–the smaller a person is, the higher on the wall he’ll sit while the ride is spinning.
You can use this same principle to separate different sizes of RNA in the ribosome. You put some RNA in a liquid, spin it around, and then find out how high up on the wall it stuck. A Swedish chemist named Theodor Svedberg figured this out sometime in the 20th century. (I wonder if he went on one of these rides before going into the lab one day? I kinda hope he did). He spun RNA around and then numbered the places that it stuck to the wall. The lower the number, the higher up on the wall it stuck, and therefore the smaller the size. Naturally, he named these number units after himself, so now we have the strangely named “Svedberg unit” (hee!) to measure RNA. We abbreviate the Svedberg as S (because spelling “Svedberg” is hard).
Therefore, when you see a number followed by “S,” it means that you can tell the size of that RNA. For example, eukaryotic RNA is broken up in to a large subunit, which is 60S, and a small subunit, which is 40S. The 60S means that the larger subunit sunk down to the 60 mark in the spinning tube, while the smaller subunit only sunk down to the 40 mark. Make sense?
Now, of course we can break up the subunits of RNA into smaller and smaller bits. So we do. In bacteria (prokaryotes), the RNA is made of two subunits: the 50S and the 30S. We break up the 30S subunit into tiny, bite sized pieces because it’s easier to deal with that way. A long time ago some super smart scientist realized that a small portion of the 30S subunit was highly conserved, and could be easily used to tell species apart. This is called the 16S rRNA in prokaryotes, and the 18S rRNA in eukaryotes. It’s used all of the time, and there has been a lot of study on these regions, so most scientific studies use this in some way.
So I am, too. Since I want to be able to tell species of bacteria apart, I chose to use the 16S rRNA region to amplify and look at for my study. This is really nice, because there are primers out there that will amplify this region very easily. Ah, the perks of looking at a well-studied bit of RNA!
But I need to have enough RNA so I can look at it, and RNA is tiny…especially when I’m talking about just the 16S region. What’s a girl to do? Amplify!
PCR is the amplification of regions of DNA or RNA. (Am I repeating myself? Probably). Knowing the properties of DNA/RNA allows us to target specific regions (like the 16S region) and selectively amplify that region alone. Step one: break up double strands. Remember how to do that? Yep, heat (go over those double bonds above if you forget). As luck would have it, we know the melting temperature of DNA and RNA (due to calculations of CG content), and so if we heat up our sample to around 94 C, those bonds will rupture and we’ll be left with single stranded DNA. (For simplicity, I’m going to talk about DNA from here on out, but the same process holds true for RNA).
Once we have our single strand, we need to focus in on just the region we want (like the 16S region). To do that, we need to tell the DNA what to replicate, and then give it the means to do so. We do this by using enzymes and primers.
Enzymes are proteins which speed up reactions without being consumed themselves. The most important enzyme in a PCR reaction is called Taq polymerase (you know it’s an enzyme when you see the -ase suffix at the end of the word). A polymerase is an enzyme that attaches molecules together (and we just so happen want to have many nucleotides attached together, so it works out for us).
Every cell that has DNA (so, pretty much every cell ever) has its own polymerase that takes care of replication of DNA and of translating bits of DNA to do work in the cell. PCR uses a polymerase from a species of bacteria, Thermus aquaticus, which normall lives in hot springs.
Have you been to any hot springs? They are ridiculous. I heard a story once about someone who jumped in one at Yellowstone national park. The meat fell off his bones before he was able to resurface. That’s stupid hot. Anyhow, bacteria are able to survive in these conditions, and do quite well, thankyouverymuch. Why am I telling you this? Well, cells that live happily at lower temperatures have enzymes that work perfectly at lower temperatures. If the temps get too high, the enzymes denature and no longer work. When we run PCR, we first start out with that melting step where we raise the temperature to break apart double stranded DNA. If we use enzymes in the PCR reaction that are denatured during that step, we either can’t continue, or we have to add more enzyme after we cool the reaction down. This is EXACTLY how PCR used to work–some poor grad student (because you KNOW professors weren’t in the lab doing this for hours on end) would have to add enzyme every 3-4 minutes during a reaction, all day. Talk about a crappy job!
So after a few years of having to manually add more and more enzyme every PCR cycle, someone thought “you know, there’s gotta be a better way!” Necessity is the mother of invention and all that, and some brilliant soul thought that there must be DNA polymerase that is stable at high temperatures. Sure enough, our friendly, heat-loving bacterium saved the day, and gave us a polymerase that doens’t denature at 95 C. Because it came from the bacterium Thermus aquaticus, we now call it Taq polymerase.
Aright, so now we have our sample of DNA, heat to break those double strands apart, and an enzyme that is stable during hot spells that will facilitate copying of regions of DNA. Now it’s time to tell the polymerase which region we want to copy!
We do this by using what are called primers. Primers are short bits of DNA that selectively attach to certain regions. Scientists design primers to attach to the parts of the DNA on either side of the region we want to amplify:
These primers are simply short bits of DNA that attach to the 3′ end of the single stranded DNA. These primers attach to the regions we’re after, and form stable hydrogen bonds. Of course, we can’t do this at the high temperature we used to break apart the DNA, so we have to cool the reaction down to 45-55 C for the primers to attach. We call the the annealing temperature and the annealing step. The exact temperature needed depends on how big your primers are, and how many Cs and Gs are involved.
The longer your primer is, the less likely it is to accidentally attach to random regions of the DNA, but the more likely it is to miss the region you actually want to amplify (long primers take a lot of time to attach, and we may not give them enough time). If you want a primer that is very specific, you design one that is really long (since it won’t attach to any other region of the DNA just by chance). If you want a primer that is sensitive, however, you design one that is shorter (since it will defiantly get the region you’re after, even if you don’t give it a lot of time). Therefore, when scientists design primers, they have to think about how specific and sensitive they want their primers, and how many mistakes they’re willing and able to put up with (called “noise”).
They also have to consider the GC content, due to those pesky triple bonds. The higher the GC content in a primer, the higher your annealing temperature. As a general rule of thumb, you want to have an annealing temperature about 5 C below the melting temperature of your primers.
Primer design is considered a little bit of science, and a little bit of art. Designers use published DNA sequences to choose good primer sites, then send off to specialized companies that make the primers for them. This is why you often see people using well-studied areas of DNA or RNA for research–the primers are already in existence, you can probably buy them in bulk, and there are certain regions that are found in all living things so you can use what are called “universal primers” to amplify DNA even in species you haven’t identified. Another plus for using the 16S region in my work!
So, we’ve broken apart the double stranded DNA, gotten our heat-resistant polymerase ready to make more DNA, and found our primers to tell that polymerase where to do its work. Now what?
Well, now we let nature take its course. We supply the Taq polymerase with all the tools it needs to do its job: the perfect environment (PCR buffer that puts everything at the optimal pH and the perfect temperature), a DNA template (our sample DNA which we broke apart), a bunch of nucleotides (in lab manuals this is called dNTP, and is really just a bunch of As, Ts, Cs, and Gs), and enough time to get the job done. We provide this in the extension or elongation step, where we raise the temperature to around 72 C (which is optimal temperature for Taq polymerase) and let it do its work. The enzyme takes all those free-floating nucleotides and lines them up all nice and neat on the template DNA.
Depending on how long our target site is, we give the polymerase 1-3 minutes to do its job (the longer the site, the longer it’s gonna take to copy it, naturally). We then repeat the process 30-40 times. After we’ve repeated it that many times, we do a final extension step at the very end, just to give the polymerase some extra time to copy all the remaining single stranded DNA (usually 7 minutes does the job nicely), and then we cool the whole reaction down to refrigerator-type temperatures to hold the DNA until we’re ready to use it.
Notice how I keep saying we change the temperature of this reaction to do the different steps? We have to have precise control over the temperature to make sure everything happens in the correct sequence. (After all, what happens if we try to copy regions of the DNA before the primers are attached? Or before the DNA goes from double stranded to single stranded? Anarchy, I tell you! Actually, the reaction just wouldn’t work. Whatever). We control the temperature by doing this entire reaction in a piece of lab equipment called the thermocycler.
We put all of our PCR reaction stuff in tiny PCR tubes…
…which we then place in the thermocycler and press the “go” button. Ah, automation at its best!
So here are the steps of PCR in a nut shell:
1. Put all the ingredients in a PCR tube: DNA, Taq polymerase, nucleotides (dNTP), buffer, primers
2. Place the tubes in the thermocycler and press “go”
3. Denaturation step: the thermocycler raises the temperature to 94 C for 20-30 seconds to melt the hydrogen bonds between the double strands of DNA and create single strands of DNA, ready for copying.
4. Annealing step: the thermocycler lowers the temperature to 45-55 C for 20-40 seconds to allow the primers to find the area on the DNA we want to amplify and attach.
5. Elongation/Extension step: the thermocycler raises the temperature to 72 C for 1-3 minutes (1 minute if the target sequence is under 500 bp long, 3 minutes if it’s over 500 bp long) and Taq polymerase goes to work copying the target sequence.
6. Repeat steps 3-5: the thermocycler then starts from the beginning again, raising the temperature to break apart the newly formed DNA, lowers the temp to anneal the primers, and raises the temp to elongate the DNA. Each time it goes through steps 3-5 it’s called a “cycle,” and the thermocycler is programed to run 30-50 cycles.
7. Final elongation step: after 30 or so cycles, the thermocycler raises the temperature to 72 C for 7 minutes just to make sure all the left over single strands of DNA have time to be copied.
8. Final hold step: the thermocycler lowers the temperature to 4-15 C (4 C is about what your refrigerator is at to keep your milk cold) to keep the DNA fresh until you’re ready to use it.
Here is a good video that goes through the whole process: PCR on YouTube
How neat is that? And so simple! Of course, I’m saying that after writing 3700 words to explain exactly how it works, but whatever. It’s simple.
By the time PCR is finished, we’re left with 1 billion identical copies of our DNA. That’s enough to do whatever we want! And you know what we want to do with all those copies of the 16S region? Pyrosequencing!
Ok, now we know a bit about DNA, replication, and PCR. The next bit of information we want about all of this is what is the exact genetic code for various regions of DNA. This information can tell us a lot about the organism from which it came, their relationship to other organisms in the world, and even the presence of mutations within particular genes. In short, knowing the actual sequence of a strand of DNA opens up a whole world of possibilities for scientists.
I want to know the particular genetic code so I can identify the bacteria I’m working with. Sure, I could use various other techniques to identify my bacteria, but conventional laboratory methods take a lot of time (as in weeks), and I have other things to do. Instead, I could extract my DNA, take a couple of hours and amplify the 16S region, and then load it all into a pyrosequencing machine and have my identifications by the end of the day. The other up side to this method is my ids are positive beyond a shadow of a doubt. No one questions identifications by genetic methods. This is why DNA is so powerful as evidence.
So, how does pyrosequencing work? I must say, this technique is brilliant…I was super excited the first time I learned about it (also? I’m a bit of a nerd). It’s a method based on sequencing by synthesis, and takes advantage of some byproducts of DNA replication. It then uses enzymes from various organisms to show us (well, technically a computer) our sequence.
Remember how I talked about how DNA polymerase facilitates the addition of nucleotides to a DNA strand? (No? Look a few hundred words above this and you’ll find out). Well, what I didn’t mention at that time was that when it does this, that reaction has a byproduct: pyrophosphate.
This is a molecule of phosphorus and oxygen that can be used to make ATP (energy). So every time a single nucleotide, no matter which one, is added to a strand of DNA, this molecule is produced and sent out into the environment.
Now, if I happened to mix pyrophosphate (abbreviated PPi so I don’t have to type that long, hard-to-spell word again) with adenosine phosphosulfate (APS), I can get ATP. Isn’t that neat? So all I need to do is make that reaction happen–can you guess what I need to do that? Yep, and enzyme.
That enzyme is ATP sulfurylase, and converts PPi from nucleotide incorporation to ATP. That newly formed ATP goes floating off into the environment, all primed and ready to do some work.
We wouldn’t want to disappoint ATP, now would we? Nope, so we give it some work to do. We have provided this newly formed energy with a reaction to run: the conversion of luciferin into oxyluciferin. Why is that important work to do, you ask? Because this reaction causes a pretty glowing light–like in fireflies!
Notice the word “luciferin” conjures up images of fire and brimstone. That’s on purpose–it’s supposed to remind you of something fiery and burning…that’s how you remember that it’s a substance that glows. Many animals in the world use this reaction all the time…fireflies are just one type. They take a substance we call luciferin, add some energy and the enzyme luciferase and cause a beautiful glow on summer evenings.
Well, some brilliant scientist thought this was neat, and decided to bring the luciferin/luciferase combo into the lab. So during the pyrosequencing reaction we take newly formed ATP and give it to the luciferase enzyme, which turns luciferin into oxyluciferin, causing it emit light.
Why do we want it to emit light? I’ll tell you in a minute. Stay tuned.
Once all the reactions are finished, we want to clean up our solution so we can run the next cycle and continue on our path to sequencing DNA, so we put in a clean up enzyme in the form of apyrase, which degrades any excess nucleotides that are floating around. This leaves us with a nice, clean slate (or, more specifically, a nice clean solution).
Alright, so in order to run a pyrosequencing reaction we need some DNA, several enzymes, and some luciferin to make glow. Lets see how those things work together to give us some information, shall we?
Step 1: Put the DNA you want to sequence in a tube (this DNA usually consists of a bunch of PCR product, so you have over a billion copies of the target sequence–the more copies, the easier it is to sequence your DNA) along with primers for your sequence (so you can get synthesis started), DNA polymerase, ATP sulfurylase, luciferin, luciferase, and apryase. This gets put into the pyrosequencing machine so the computer can take over.
Step 2: Press “go” on the machine. The computer adds one of the four nucleotides (A,T,C, or G) at a time–let’s say it starts with A. It floods the tube with the A nucleotide, and the enzymes take over.
Step 3: Your DNA strand is primed and ready, so if the first nucleotide on the template strand is a T, then the A that just flooded the solution will be added by DNA polymerase on the new complimentary strand. (If you forget how DNA replication works, check out my other blogs, or watch this video).
Step 4: The incorporation of that A into the new strand causes PPi to be released. That PPi is taken by the ATP sulfurylase and converted into ATP.
Step 5: The ATP is used as an energy source to allow luciferase to turn luciferin into oxyluciferin, which emits light.
Step 6: The light given off by the reaction is recorded by a camera attached to the computer, and logged as a peak called a pyrogram.
Step 7: Once DNA polymerase has used up all the A nucleotides it needs, apyrase degrades all the extra nucleotides floating around and gets the solution ready for the next one.
Step 8: The computer floods the solution with another nucleotide (let’s say G), and the process starts again.
If there is more than one identical nucleotide in sequence (say GGG or TT), then more PPi is released (if there are two nucleotides in a row, then twice as much PPi is released; if there are three then 3x as much is released, etc.). More PPi means more ATP. More ATP means more luciferase action. More luciferase action means a brighter light. A brighter light is recorded as a higher peak by the computer.
The above steps are repeated until the entire sequence of DNA has been replicated. The computer then looks at they pyrogram and translates the peaks into a DNA sequence.
Proteins denature under heat stress. This denaturation of proteins causes the transcription of heat shock proteins (HSPs) to deal with the problem. The production of HSPs is rapid, and facilitates repair of the damaged cell. If the temperature remains high, heat shock proteins will remain at a steady-state level in the cell, which is higher than the initial, cooler temperature state, but lower than the peak reached when cell repair was active. The cell has a need for several HSPs at all temperatures, but the need is elevated at higher temperatures and results in an increased rate of synthesis.
There are two major classes of heat shock proteins: proteases (Lon) and chaperones (DnaK, DnaJ, GrpE, and GroELS). Proteases degrade proteins that are misfolded (and some normal proteins) while chaperones recognize exposed hydrophobic regions that shouldn’t be exposed, binds to them, and places those proteins in a chamber which allows the proteins to refold properly.
HSPs are grouped into families based on their molecular weight. Proteins with a weight of about 70 kDa are grouped into the Hsp70 family. Those with a molecular weight of 60 kDa are grouped into the Hsp60 family, etc.
Following a temperature upshift (ex: from 30-42 C) there is an increase in the amount of sigma factor 32, or RpoH. This factor is responsible for the synthesis of at least 30 HSPs that work in the cytoplasm. Sigma 32 is not active at lower temperatures, and becomes stable after heat shock. Because of this, sigma 32 is considered a major heat shock regulon. Organsims that don’t make sigma 32 are unable to grow at temperatures above 20 C.
During normal temperatures and growth, sigma 32 is an unstable protein with a half life of 60 seconds. After a heat shock (e.g. 30-42 C) the protein stablizes for a few minutes, and it accumulates in the cytoplasm. At non stress temperatures, cytoplasmic proteins DnaK and DnaJ bind to sigma 32, making it subject to proteolysis by proteases (including Lon). At higher temperatures, DnaK and DnaJ preferantally bind to denatured proteins, leaving sigma 32 unmolested and able to bind to RNAP. This sigma-RNAP complex protects sigma 32, and results in a holoenzyme that transcribes the Hsp sigma 32 regulon. Therefore, it is the amount of denatured protein (as opposed to temperature directly) that results in the transcription of heat shock proteins. (This point is further supported by the transcription of HSPs after other types of damage that cause protein denaturation). During heat shock periods, there is an increase in the transcription of mRNA for sigma 32. After heat stablizes, sigma 32 activity lowers rather than the concentration.
At very high temperature (45-50 C) the sigma E regulon is activated, which protects extracytoplasmic proteins from damage. Very high temperatures can cause proteins in the membrane to misfold, which is the signiling pathway to activate sigma E (or sigma 24) in the cytoplasm. When sigma E is activated it binds to RNAP, and the sigma E regulon (which consists of at least 11 genes) is transcribed. (This mimics the way sigma 32 is controlled and transcribed) These genes code for proteins and proteases that are involved in the folding, refolding, and degredation of misfolded proteins in the cell envelope. At lower temperatures, sigma E is bound by an anti-sigma factor in the inner membrane. Envelope stress allows for the release of sigma E, when then binds to RNAP.
Sigma S is considered the master regulator for general stress response, including heat shock, nutrient stress, etc.
When damage to DNA is sensed, the SOS signal induces over 20 unlinked genes. These genes cope with the DNA damage by remairing the damaged DNA, allowing DNA replication to proceed past the damaged site (translesion synthesis), and stalling cell division to allow time for DNA repair. Normally, the SOS response system is suppressed by down regulating the SOS genes. This process is governed by a master repressor protein called LexA.
Damage to DNA is dected when DNA polymerase encounters a lesion during DNA replication. This causes replication to stop, which leaves a tract of single stranded DNA exposed without being replicated. Since ssDNA is prone to attack, the cell covers the exposed DNA with a protein termed RecA, which starts binding at the 3’ end. The RecA-DNA complex causes LexA to bind and cleave. After enough LexA is broken, the repair genes are expressed. SOS genes have LexA boxes at their promoters. The LexA boxes have slight differences in sequence, and therefore slight differences in their affinity for LexA. Genes with weak affinity for LexA get expressed earlier than genes with strong affinity for LexA, thereby allowing a cascade of gene expression based on the amount of LexA present (which is directly tied to the amount of RecA-DNA complex, which is directly caused by the amount of single strand DNA exposed, which is a measure of the DNA damage).
Some of the earliest genes expressed in response to damage are the uvr genes, which encode for nucleotide excision repair (NER). These proteins detect damage on a single strand of DNA, and excise the damaged area, along with bases on either side of the damage. The gap is filled in by DNA polymerases, which leave the DNA intact and replication can continue. Most lesions are fixed in this manner.
If NER is insufficient to repair the damage, then RecA carries out recombination. The RecA-DNA complex formed at the first sign of damage catalyses the pairing of ssDNA with duplex DNA, one strand of which is homologous to the ssDNA. Once the homologous tract is found, RecA will facilitate strand exchange.
If even recombination doesn’t work, the umuDC operon is expressed, which restarts replication at the stalled fork by employing mutagenesis. PolV, a special DNA polymerase, is encoded by the umuDC operon, and it inaccurately replicates DNA over the lesion. It may introduce incorrect nucleotides, and with them mutations. PolV is formed when the UmuD protein is cleaved by RecA, and associates with UmuC, thus forming PolV. After PolV places a few nucleotides, it is replaced by the accurage PolIII DNA polymerase, and replication continues as normal until another lesion is encountered.
The whole process of DNA repair must happen before a cell divides, and therefore cell division is delayed by the SOS protein SulA. This inhibits the function of proteins involved in cell division, including FtsZ (the septation protein). Once DNA is repaired, Lon protease can use SulA as a substrate, breaking down SulA and allowing the cell to septate as normal.
After DNA repair is accomplished, LexA concentration must be restored to shut off the SOS network. This is done by a number of proteins, one of which is called DinI. DinI structure resembles DNA, and this allows DinI to interact with the RecA filiment. This interaction inhibits both the recombinase and protease activity of RecA. Once RecA is inhibited, it no longer breaks down LexA, which begins to build up in the cell again, and once again works as a master repressor. There are other proteins involved in the shut off of the SOS network (such as RecX) but many of these have not been well studied.
Hello again! Today I’m going to tackle a broad, essay type question that pertains to bacterial movement: Chemotaxis.
Chemotaxis is the ability of a bacterium to move along a concentration gradient, either towards an attractant or away from a repellent. The attractant or repellent is termed a chemoeffector, and is monitored by a system of transmembrane sensor proteins, called methyl-accepting chemotaxis proteins (MCP), or receptor-transducer proteins. These proteins affect a two component system: CheA, a cytoplasmic histidine kinase, and CheY, a response regulator. Action upon this system affects the flagellar motor.
Bacteria swim by rotating flagella. Counter-clockwise rotation align the flagella in a single bundle, causing the bacterium to swim in a straight line (termed a “run”). Clockwise rotation causes this flagellar bundle to break apart, and results in random tumbling in place (termed a “tumble”). As few as 25% of the flagella need to rotate clockwise to cause random tumbling, but the more flagella rotating in this manner, the greater the change of direction.
Bacteria are unable to choose the direction in which they swim, and are unable to swim in a straight line (a run) for very long due to rotational diffusion; they “forget” which direction they were going. This results in random run and tumble movement across space. Chemoeffectors influence this random movement. When a bacterium senses it is going towards an attractant or away from a repellent (the “correct” direction from the bacterium’s point of view), it will swim in a straight line for longer; this results in a longer run vs tumble phase. The presence of an attractant decreases the probability of clockwise rotation of flagella, keeping the bacterium from tumbling. The presence of a repellent increases the probability of clockwise flagellar rotation, resulting in a shorter run, and more change of direction. Therefore, attractants see longer, more frequent runs mixed with shorter, less frequent tumbles, resulting in an overall movement towards the attractant (or, conversely, away from the repellent).
Bacteria sense chemoeffectors on a temporal gradient: they are able to remember past concentrations long enough to compare them to present concentrations, and then use this information to make a decision. This memory is long enough for the bacteria to compare two points more distal than its body length, yet short enough to signal the bacteria before it tumbles randomly.
Six genes are required for chemotaxis: CheA, CheB, CheR, CheW, CheY, and CheZ. In mutants that have any of of those genes knocked out, chemotaxis is impossible. As mentioned above, chemotaxis is controlled by a two-component system, which is alerted by methyl-accepting chemotaxis proteins that span the membrane and monitor chemoeffectors in the periplasmic space. CheY is ultimately responsible for the way in which a flagellar motor turns. If it attaches to proteins in the flagellar motor (FliM), then the motor will turn clockwise. If it doesn’t, the motor turns counterclockwise. Therefore, CheY must attach to the flagellar motor to cause tumbling. CheY is activated by accepting a phosphate group from CheA. CheA is signaled by transmembrane proteins, of which there are 5: Tar (taxis to aspartate and away from repellents), Trg (taxis to ribose, glucose and galactose), Tap (taxis to dipeptides), Tsr (taxis to serine and away from repellents) and Aer (taxis to oxygen as it oxidizes FADH to FAD). The presence of these substances in the extra cellular space causes a conformational change in the transmembrane protein. This initiates a CheW mediated response in CheA phosphorilation:
CheA + ATP=CheA-P + ADP + Pi
CheA then phosphorilates CheY: CheA-P + CheY=CheA + CheY-P
The binding of CheY-P to the flagellar motor causes clockwise rotation. If CheY-P is dephosphorilated, then it will not bind and the flagellar motor will turn counter-clockwise. CheZ is responsible for the dephosphorilation of CheY-P in the cytoplasm. Under normal circumstance, CheY is phosphorilated and dephosphorilated at a constant rate, allowing for the random run/tumble action observed in bacteria not experiencing chemotaxis. When an attractant is sensed, autophosphorilation of CheA is decreased, which decreases the phosphorilation of CheY, and therefore the probability of flagella turning clockwise. When a repellant is sensed, the exact opposite happens.
CheA also phosphorilated CheB, which is a regulator that governs adaptation to a particular concentration of attractant. CheB-P removes methyl groups from glutamate residues in the receptor-transducer proteins. CheR adds methyl groups to the glutamate residues. When an attractant is present, CheA does not become phosphorilated, and therefore CheB does not phosphorilate either. CheB cannot remove methyl groups from the glutamate residues. Higher methylation of glutamate residues stimulate tumbling, and therefore the chemotaxis stops for this concentration of attractant. When the concentration changes again, chemotaxis returns as normal.
Welcome Back! A year of research under my belt, and I’m back to answer some more GRE questions. Here’s today’s:
An infectious agent that appears to have no nucleic acid is a: A) bacterium B) bacteriophage C) viroid D) virus E) prion
This is one of those basic fact-knowledge questions, although even if you don’t know the exact properties of everything on this list, you may be able to figure it out. So let’s get into what the question is asking.
What exactly is an infectious agent? An infectious agent is some sort of organism (we can debate whether a virus is a true organism later…) that invades another organism to utilize the host’s resources to some end. Pop culture types call these things “germs,” which is a term that encompasses anything that makes humans feel bad in some way.
Scientists have the annoying ability to break up these germs into like groups that act and infect in a particular way, which usually involves the anatomy, physiology and phylogeny of the organism. This question is basically testing your knowledge of microbial anatomy.
On to the second part of the question. What is nucleic acid? This is the “NA” portion of DNA or RNA. The D and R simply refer to the sugar groups attached to the nucleic acid. Most organisms have some sort of nucleic acid (either DNA, RNA or both) that they use to reproduce. After all, DNA is the blueprint for life. I say “most organisms” because as we get better and better at finding things, we’re noticing an exception to this rule. More on that in a moment.
Alright, so now that we know what the question is asking, how do answer it? This is simply a fact question, so you need to know a little bit about these organisms. Let’s start with bacteria.
Bacteria are single celled organisms that have a cell wall and the ability to live free. Most people know about bacteria after a trip to the doctor and a filled prescription for antibiotics. The main thing you need to know, though, is these organisms reproduce by DNA, and therefore have nucleic acid.
Viruses are also single celled organisms (although here is where every biologist begins the “what is an organism?” debate) that is rather simple–they consist of some proteins and DNA or RNA. In order to do any biological process, they must invade a host cell and take over the cell’s functions. So, while simple, these still have nucleic acid, else they wouldn’t be able to take cells hostage.
Bacteriophages are much like viruses–in fact, they are a form of virus that infects specifically bacteria (hence the “bacterio” portion of the name). Just like other viruses, these are made of protein and DNA or RNA, and they take over a cell’s function inorder to reproduce. Did you catch that? They have nucleic acid.
Viroids are along the same line, and just like the name suggests, are a lot like viruses. These are specifically plant pathogens, consisting of circular RNA and protein. Once again, an organism with nucleic acid.
That leaves prions. This is one of those things I alluded to earlier–a new form of what may be an organsim or may not, that causes infection. A prion is misfolded protein that is able to act as an infectious agent. That’s pretty much all we know about it now. We don’t know how it reproduces (because it doesn’t have DNA or RNA!) nor do we know how it forms. There has been a lot of researhch into prions lately, as people are beginning to think these buggers are responsible for a variety of nasty diseases. The biggest problem is because we have so little knowledge about the prion, we don’t know how exactly they are spread, or how to stop them. Ah, the mysteries of medicine.
Today’s question involves how cancerous cells divide and actually become tumors. Here’s the question:
All of the following mechanisms have been shown experimentally to the contribution to the formation of cancer cells EXCEPT: and then there were answers.
The basis of this question is the nature of cancer. Cancer is the abnormal growth (and possible spread) of cells. These cells can invade and destroy other, healthy tissues, which causes the pain and death of cancer. The question, then, is what actually causes cancer to develop?
Since its discovery, scientists have been studying the mechanism of cancer. Their experiments have been focused on both the destruction of cancerous cells, and the mechanisms needed to cause normal cells to turn cancerous. There are several ways your normal tissues can cause you problems:
1) DNA becomes damaged. All cells (with the exception of mature red blood cells) have a nucleus, and this nucleus contains the blueprint for the cells. If the portion of the blueprint that tells the cells when to stop producing gets damaged, then the cells keep dividing with no limit. That causes tumors.
2) Growth factors. Growth factors are natural proteins that stimulate reproduction and differentiation. The presence or absence of growth factors accounts for how often the cell reproduces.
3) Introduced DNA. Viruses have the annoying tendency to inject their DNA into other cells. While some viruses compeletly take over the cellular functions, some just incorporate themselves into the host DNA and for long periods of time. This foreign DNA can cause cells to react strangly, and can cause cancerous cells to develop. (Heard of the shot developed to prevent cervical cancer? That works on this basis).
These are three major factors that determine if a cell is going to turn cancerous. Now, back to our question:
All of the following mechanisms have been shown experimentally to the contribution to the formation of cancer cells EXCEPT:
A) Abnormally high energy reserves in cancer cells that cause them to divide too quickly B) Mutations that cause excess production of growth factors by cancerous cells C) Mutations that inactivate genes that normally inhibit cell reproduction D) Mutations that reduce the need for growth factors in cells E) Viruses that carry genes that transform normal cells into cancer cells
Which one of these wasn’t mentioned above? A. There we go!
Well, we’re back in business! I’ve decided to focus a bit on microorganisms, since school has started again and I need to bone up on my micro before I have to lecture about it. In light of that, here’s today’s question!
In E. coli, induction of the lactose operon occurs when allolactose binds to:
This question tests your knowledge of how gene expression works, but tries to confuse you by giving specifics about E. coli. First things first–how does gene expression work?
Genes are some of those super complex things in biology–and one of those things that has a million names attached. Fun for us! Anyhow, sections of DNA code for proteins. They do this by creating messenger RNA (or mRNA) that tells the cell what to make. However, the DNA doesn’t just randomly make messenger RNA and code for proteins…specific conditions must be met for this to happen. Of course this causes things to be much more complex.
DNA has specific sequences that tell the cell when to make mRNA, when to stop making mRNA, and when to prevent mRNA from being made. All these sequences together with the actual genes are called the “operon.” Often time, in addition to the operon is a regulator gene called the repressor or co-repressor that allows the operon to be turned on.
If a repressor is present, it usually stops the gene from being expressed (keeps the proteins from being made); hence the name “repressor.” Certain criteria must be met for the repressor to allow gene expression.
So, lets look at E. coli specifically: this bacterium loves lactose (anyone lactose intolerant? You know that horrible feeling you get after drinking milk? That’s because the E. coli in your gut loves the undigested lactose and poops out acid and gas. Thanks microbes!) and therefore has a gene dedicated to breaking down this sugar. This gene is one of those with a repressor, however. It wouldn’t make much sense for E. coli to try and break apart lactose if there wasn’t in the environment, now wouldn’t it? When lactose is present, a metabolite of lactose called allolactose is present. Allolactose binds to the repressor, which then allows the gene to be expressed. If there is no allolactose in the environment (and therefore no lactose) the repressor stays in effect and keeps the gene dormant.
Well, back to our question:
In E. coli, induction of the lactose operon occurs when allolactose binds to:
Since we know that induction means “get started” and an operon is that group of DNA that includes the operator, promoter and genes, we can now answer the question. The answer is “E” the repressor. Yay!
Well, vacation is over, and now it’s time to get back to the great subject of biology! Let’s jump right into it, shall we?
The addition of colchicine to a culture of actively dividing flagellated eukaryotic cells inhibits all of the following except: (And then there’s a list of answers).
Well, I chose kind of a pain in the ass question, didn’t I? The GRE loves throwing specific substances/structures/species at you to determine if you know what it is or not. That’s fun and all, but what if you have no idea what the bloody thing is? First, I’m going to tell you all about this colchicine stuff and its effects on cells. Then I’ll give you some tips to make guessing the correct answer easier.
So, what is going on with this question? First off, notice that the test writers are doing that thing where they fill the question up with lots of multi-syllable words that may or may not confuse the reader. Don’t let them win! The two biggest misleading words in this question are “flagellated” and “eukaryotic,” neither of which have much to do with the meat of the question, which is “what the heck is colchicine?” If you find yourself getting bogged down in the complexity of the question, just take a moment to define each of the words and decide if they actually have any affect on the question itself. In this case, “flagellated” (the state of having a flagellum, or thing, whip-like tail used to propel and organism) really just gives you more detail about the cells the colchicine is being dumped on, while “eukaryotic” (cells with membrane-bound organelles) gives you even more detail. Fun! Ignore them for now.
Now to the real question: what is colchicine? Colchicine is an organic compound (molecules that contain carbon) that also contains nitrogen as its key component. This particular nitrogen containing organic compound (or “amine” for short) is produced by the Autumn Crocus, a very pretty little plant that you really don’t need to know about. What you do need to know is that colchicine is very poisonous, and is therefore used therapeutically by doctors around the globe.
Colchicine causes vomiting and defecation in humans, and is therefore prescribed to combat joint issues such as gout. (For those who don’t know, gout is a very painful condition in which uric acid crystals form in the joints of the lower extremities. This condition is related to kidney stones, and flare-ups happen after intake of rich food and drink. It was known as a disease of the rich in days of yore, but is now known as a disease of the unlucky and limping).
On a cellular level, colchicine inhibits the formation of microtubules. It does this by inhibiting tubulin–the substance responsible for making microtubules.
Microtubules are exceptionally important in two areas: growth and structure. Microtubules make up the main structure of the cytoskeleton, which gives the cell its shape. No microtubules, no cytoskeleton. That’s a bad thing for new cells. Microtubules are also important during mitosis.
Have we gone over the stages of mitosis yet? I don’t think so–that’s a long lecture so I’m avoiding it. Well, the short version is mitosis is the process by which a cell reproduces itself. There are several stages of mitosis, during which particular things happen including the copying of DNA,and the relocation of the genome to the new cell. Microtubules are responsible (in the form of spindle fibers) for pulling the DNA from the center of the mother cell into the new daughter cells. If no microtubules form, then the DNA cannot migrate to the new cell, which means no new cells. Growth is inhibited.
This inhibition of growth makes colchicine a great drug for fighting cancer cells. The hallmark of cancer is its unfettered reproduction; since colchicine stops reproduction, flooding cancerous cells with colchicine stops their growth. Good! Of course, it also stops the growth of any healthy cells it touches, so it is only used sparingly and is not a miracle cure.
Ok, now we have an idea as to what colchicine does. Lets get back to the question:
The addition of colchicine to a culture of actively dividing flagellated eukaryotic cells inhibits all of the following except:
A) Movement of flagella
B) Growth of flagella
C) Formation of mitotic apparatus
D) Formation of microtubular cytoskeleton
E) Polymerization of tubulin
The answer seems obvious, right? Hopefully? Since colchicine inhibits tubulin which therefore inhibits microtubule production, all growth is stopped (due to lack of spindle fibers), formation of mitotic apparatus is inhibited (due, once again, to lack of spindle fibers), the cytoskeleton is stunted, and polymerization of tubulin is halted. Basically, everything involving growth and reproduction is stopped. However, this substance doesn’t have any effect on already formed microtubules–you see, it only stops the substance that makes up new microtubules, it doesn’t break down old tubules. So movement and function of mature cells goes untouched. Movement of flagella, therefore, is unaffected by colchicine. The answer is “A.” Yay!
So what happens if you’re sitting at the test and have no freakin’ clue what colchicine is? You may be able to figure it out with a little bit of effort. Look at the answers given here:
A) Movement of flagella
B) Growth of flagella
C) Formation of mitotic apparatus
D) Formation of microtubular cytoskeleton
E) Polymerization of tubulin
The great thing about a multiple choice exam is the answer is staring you in the face–you just have to recognize it. In this particular example, the question is asking the effects of some substance on some cells. Your first task is to break down the question to its essential parts. Don’t go trying to answer a question that isn’t even asked! So, what are the effect of this substance? Look at the answers–two of them (B and C) are directly related to the growth and reproduction of a cell. Formation of the cytoskeleton has to do with growth as well, and polymerization is a fancy word for “making” or “putting together” or “growth.” So 4 of the 5 answers have to do somehow with growing. Whenever you see a link between most of the answers, and the question asks “which is not like the other” then you have a pretty good idea of the answer. Make sense?