Tag Archives: molecular motors

Reading your Blueprint: DNA Fingerprinting

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Who are you? That’s the question asked repeatedly in the song by The Who that CSI uses as it’s theme song. The answer, it seems, is found in the various pieces of evidence the Crime Scene Investigators find. Sometimes it’s pieces of fiber or pollen that eventually lead to the answer, but by far the most common method of determining identity is analyzing DNA evidence. What you see in the show is attractive people in lab coats taking samples of tissue, sticking them into some machine and then looking at a bunch of bars on a lit screen and saying things about what they mean. How is this any different from the old methods of reading patterns in tea leaves or goat entrails?

First off, I should probably point out that tv shows don’t always get things right. It usually takes much longer to get a result from a DNA sample, and if you recall the Casey Anthony trial and the OJ Simpson trial, DNA evidence does not always lead to a sure result. Well, what good is it, then? It’s not that DNA evidence isn’t useful, it’s just that there are circumstances where it isn’t available or isn’t clear. Similar to fingerprints.

So how do they analyze the samples they gather? They can’t use karyotyping usually, because they need living cells in order to do that. Thankfully, technicians don’t need to have DNA in chromosomes in order to be able to analyze it. As long as all they are looking for is straight DNA, they can get it from almost anything biological. Hair, skin, saliva, blood, and other fluids all contain viable DNA.  You still have to get it out of the sample first.

Close up of polymer gel structure. Water and small molecules can pass through spaces in the mesh. Image adapted from http://bit.ly/qvsOix

One way to do this is to crush and dissolve the sample using proteases. Proteases are enzymes that eat up proteins. They are present in a lot of different cells and even in some of the food we eat. If you’ve ever eaten a lot of pineapple and found your mouth sore afterwards, the reason is because of the proteases in the pineapple. This is also the reason why it is very difficult to make pineapple jello. Jello is a type of gel, a substance made up of proteins that form a mesh that traps water inside. If there is too much protease present, that mesh can’t form, and the Jello mix stays syrupy. The materials that keep your body’s cells from moving all over the place are also a mesh of proteins, and so if there is enough protease, some of this mesh will be dissolved. This is why pineapple can make your mouth sore. It is also why a strong protease can eat away the proteins that surround a DNA sample. Hair samples, in particular, have a lot of protein surrounding the DNA, so there needs to be a lot of protease to get to it.

Another way to get at the DNA is by using a detergent. Laundry detergents clean clothes by breaking up the oily materials that trap stains in the fabric. The membranes of cells are also made of oily material, so by using a detergent, we can wash away the cell membranes and get to the DNA inside.

Often both a protease and a detergent is used just to be sure the DNA is accessible. After this, the sample is put through a centrifuge to remove the lighter, non DNA molecules from the heavier DNA. Once the DNA is free, chemicals or heat is used to separate the two strands of DNA into single strands. This allows the restriction enzymes to work on the DNA. Restriction enzymes cut the DNA at specific areas that a technician can determine ahead of time. DNA is formed of four different bases: Adenine, Guanine, Cytosine, and Thymine, Each enzyme attaches to a certain sequence of these bases and cuts the DNA where ever that pattern shows up. DNA from different individuals will have different lengths between each recognized pattern and so will be cut up into differently sized sections after the restriction enzymes do their work.

Image from http://bit.ly/9h7sIW

The cut up DNA is then poured into a gel, usually made out of agarose or polyacrylamide. Just like Jello, these gels are made of long molecules connected together to form a mesh that partially traps water. The DNA fragments can travel through the spaces in the mesh, but not very easily. Lab workers apply an electric field to the gel using a tray with exposed electrodes that holds the gel immersed in a bath of mostly water mixed with some chemicals to help pass the electricity to the sample.  DNA is made of sugar molecules, and like most things made of sugar, it becomes negatively charged very easily.  In fact, unless DNA is in a solution with a high pH, it will have a slight negative charge, and so the electric field made by the tray device  pushes DNA down the gel toward the positive electrode in a process called electrophoresis.

http://www.biologyreference.com/Dn-Ep/Electrophoresis.html

The larger the piece of DNA, the harder it is for it to pass through the mesh of the gel. Smaller strands will therefore travel farther along the gel in a given period of time.Fragments of DNA that are the same size will be in the same area of the gel after they’ve been pulled through it, forming bands in those areas. These bands are still not visible, however, until the lab worker applies some kind of label to the DNA. This could be a chemical dye that binds to DNA, or DNA fragments with radioactive isotopes attached. The most common chemical dye is ethidium bromide, which is only visible under a black light. The lab worker can place a gel stained with ethidium bromide on a scanner attached to a black light to record the bands in the gel. In the case of radioactive isotopes, a film sensitive to x-rays can be placed on the gel which will turn dark where ever the radioactivity is.

Gels fall apart very easily, so many lab workers will transfer the DNA from the gel to a sheet of filter paper by pressing the paper to the gel and passing a current through it for some time. Then they can apply a label as before to see the bands of DNA.  These bands are what you usually see on tv shows where they compare DNA. By looking at where the bands from different samples show up, a lab worker can tell which samples are from the same source and which ones aren’t.

Debbie Knight at biologyze gives an excellent  detailed accounting of the process of gel electrophoresis here if you’re interested. She uses it to analyze proteins, but the idea is very similar.

Sometimes there isn’t enough DNA in a sample to get visible bands, in fact, this is usually the case. For these situations, there is PCR. PCR stands for Polymerase Chain Reaction. Polymerase is an enzyme that replicates DNA, but it’s more complicated than that. In order for the DNA to be replicated, it has has to be separated into single strands from its usual two stranded arrangement. This is called denaturing the DNA. In nature, this task is performed by proteins called helicases, however in the lab, heat or other chemicals can do the job.  Chemicals are somewhat difficult to work with, because they have to be washed out of a sample every time they are used. Heat is a much better denaturing agent, but most polymerase molecules can’t stand the heat needed to separated the DNA.

http://users.ugent.be/~avierstr/principles/pcr.html

Thankfully scientists found the molecule Taq, a DNA polymerase found in bacteria that can withstand the heat.  So what happens during PCR is that after Taq is added, the samples are first heated to a temperature high enough for the DNA to denature. Then the temperatures lowers, letting initiating sections of DNA called primers to attach or anneal to the fragments. Then the temperature raises again to a temperature that is ideal for Taq to replicate the DNA. Finally the samples are heated once more, so that the new DNA fragments separate again with twice as many fragments as before. This reaction is performed again and again, so that first two new strands are made, then four, then eight and so on, the amount of DNA increasing exponentially with every cycle. This way even with a miniscule amount of DNA you can have enough of a sample to work with.

 

Molecular motors

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At this very moment, inside every cell of your body, there are thousands, even millions of machines, so small that they can’t even be seen with a conventional microscope, performing tasks in mere fractions of a second without any direct supervision. There are tiny molecular robots working together inside us to accomplish goals that scientists are only beginning to understand, every moment, of every day, through out each of our lifetimes. And it’s not just in our bodies. In the bodies of every living thing these things are active. They are, in fact, required for the basic prerequisites of life, yet individually, they are nothing but dumb molecules, practically inert unless the molecules they interact with are present. These things are called molecular motors.

There are a bunch of examples of these and every time I think about them I’m amazed. I just have enough space to talk about maybe two of them, but really almost all molecular motors rely on three things: energy, tubes, and molecular components.

Chemical structure of ATP

The usual form of energy for molecular motors, the twenty dollar bill in the economics of proteins, is ATP. ATP stands for Adenosine triphosphate, and its called that because it is a molecule made up of adenosine attached to three phosphorus atoms (called phosphates while they’re still bonded to a certain number of oxygen atoms) arranged in a line. If proteins need energy for a reaction, they get the energy by lopping off one of the phosphates of an ATP. ATP then turns into ADP (adenosine diphosphate) and the energy that was stored in the bond between the phosphate and the rest of the molecule goes into whatever is needed by the molecular motor or by the reaction that’s going on. When a molecule snaps off a phosphate, the phosphate is bound to that molecule for a while and the molecule is said to be phosphorylated.

This might be a little boring in itself, but the cool thing is that so many different reactions inside a cell depend on the same ATP molecule. Almost every reaction between proteins involves a phosphorylation or a de-phosphorylation. When you eat food, what you’re really doing is supplying your body with ATP.

Adenosine is actually one example of a class of proteins called nucleotides. They can all carry phosphates, though adenesine and guanine are the ones typically used for energy. The reason nucleotides are called nucleotides though, is that they are all present in DNA, which is found in the nucleus of a cell. There are the four typical nucleotides you might remember from high school biology (Adenosine, Guanine, Thymine, and Cytosine) and then there is another that is only present in RNA, uracil. DNA forms a double helix, or a twisted ladder, with each nucleotide forming a rung in the ladder by binding with a partner nucleotide. As a general rule, adenosine binds with taurine, and cysteine binds with guanine. A goes to T and C goes to G. This is important because, by keeping to this rule, a cell can use single stranded DNA as a sort of photo negative, and use it to make the opposite strand over and over again as many times as it’s needed.

The other “tubes” come in two flavors: microtubules and actin fibers. Microtubules are the scaffolding and road system of an animal cell, keeping thing in place and providing a network of connections to every organelle. Actin fibers are similar to microtubules, except they are more temporary. They are used a lot in things like amoebas to form psuedopods and move around.

Finally there are the actual proteins that make up the molecular motor. Kinesins and myosins are perhaps the neatest looking proteins. They look like thin cartoon characters with really large feet. What they do is they carry or pull things along a tube (a microtubule for kinesins and actin fibers for myosins)by “walking” along it. You can see movies of this like the one below. The feet start out latched to a tube. Then one “foot” will release and latch on again a little ahead of where it was before. Then the other foot will to the same and so on so that the molecule is walking along the tube while carrying some cargo.

DNA polymerase is another molecular motor, though it’s often classified as an enzyme. It latches on to a single strand of DNA, takes phosphates of nearby nucleotides and then uses the energy from the phosphates to attach the nucleotide to its partner on the DNA. If the nucleotide is the wrong match, it gets thrown out. DNA polymerase is basically like a factory worker on an assembly line.

But it’s just a protein.

We’re talking atoms bound together, people. And yet they’re doing these sophisticated things. Perhaps its time we recognized our protein overlords.

Incidentally, if you are interested in this, you might want check out this blog as well: http://informedworship.blogspot.com/

Flagella and Philosophy

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Most bacteria, including E. Coli bacteria, which cause food poisoning, move around by using flagella. Under the microscope, flagella look like kinked up hairs sticking out of the cell and undulating rapidly. If you remember high school biology you might already know, or think you know all about flagella. They’re those hairl-like  things that cells whip back and forth to move forward right?

Not exactly. There are basically two kinds of flagella, the ones that bacteria have, and the ones that eukaryotic cells have. The eukaryotic ones do in fact whip back and forth. The bacterial ones, however, actually propel the cells they’re attached to by corkscrewing through the fluid. As I went over in a previous post, the situation bacteria are presented with isn’t the same as a submarine in a ocean. It’s more like a submarine in a large silo filled with vibrating pebbles. Drag is a huge deal. Bacteria need a powerful technique to move around and a corkscrew type action works rather well.

When you use a corkscrew on a wine bottle you basically turn it around in a circle as it progresses into the cork. So here’s a question: how does a bacterium do that? The flagellum has to be attached to the cell with a buttload of strength to pull the cell along, but at the same time it has to be able spin around like a drill bit. If you look at all the different components of the flagellum (pictured below) you can see that the structure is pretty complicated. WTF? Aren’t bacteria supposed to be primitive organisms?

Well, yes and no. You see, the thing that’s easy to forget about evolution is that every thing that’s around has been evolving for just as long as everything else. It’s just that while our cells were busy trying to figure out how to differentiate and work together to get to places where there was better food and water and such, bacteria were hunkering down and learning to live in the environments they found themselves in. They’re just as good at being bacteria as our cells are at being part of a larger organism. Still, the flagellum is so mind-bogglingly complicated and yet robust in its implementation, that it has from time to time been used to prove or disprove the non-existence of God.

The argument runs something like this: “We and the organisms of Earth have to have been created by a God,” say creationists, “because the bacterial flagella is so complicated that it could not possibly have been formed by chance, any more than a hurricane could blow through a ship yard and create an aircraft carrier.”

“But,” say non-creationists, “each protein that makes up the structure of a flagellum looks similar to proteins used in other, less complicated bacterial structures. Furthermore the flagellum of E. coli is only one variant of many, indicating that one Doesn’t it make sense that flagella might have come from mutations that put the proteins together in ways that were somewhat beneficial? If even one bacterium survived a trying situation better than its neighbors, it would quickly begin to dominate. And evolution of bacteria has been directly observed in nature and in the lab. At the very least that seems more plausible than everything getting poofed into existence through some unknown process by a magical man in the sky.”

You can probably tell which side I favor.

Something that you start to notice if you study biology is how complicated and diverse proteins are. Proteins aren’t just little specks that bump into each other in random ways. Each protein has a different shape and different areas that have different properties that interact with other proteins in different ways depending on environmental factors. It’s an imperfect analogy, but you could almost say proteins have personalities. Almost like people, proteins will work together, compete with each other, and even find things in their environment and use them to accomplish a “goal.” A protein may be stuck in a bad position, for example, and it may come into contact with another protein that likes to pry things apart, and then they will help each other out.

So it’s not exactly the same situation as a hurricane picking up a bunch of aircraft carrier parts and arranging them into a complete aircraft carrier. Its more like a bunch of people who don’t know how to make an aircraft carrier getting a steady supply of parts and enough time and resources to try things out. Eventually, given enough time, people in that situation will eventually make something like an aircraft carrier. Though they’d probably start out making houses, cars, and a number of other things out of the parts before hand. In fact you could look at the current real-life production of an aircraft carrier as an example of evolution. Evolution is simply the effect of variation subjected to some environmental pressure. There were many different types of ship before the aircraft carrier came about. For one thing, there had to be aircrafts in order for there to be any use for one. Humans wanted a place to put planes on the ocean and the aircraft carrier fulfilled that purpose

In a similar way, proteins just want to be in equilibrium, and so they will often use things like enzymes to make equilibrium easier to achieve.

If you are doubtful of any of this, you need only see a video of how a flagellum is formed in a bacteria. The formation of a flagellum is a marvelous and awe-inspiring dance and it happens hundreds of times a second in a single organism ALL THE TIME. You don’t need religion for miracles. You just have to look around.