What is Life?

This will be a short group-discussion project with no slides. You may have a 1-page handout.
In the beginning of the year, I told you our current definition of life. In particular, life was defined as requiring cells. Then, there were a list of requirements, such as a membrane or other device to keep the inside and outside different, and the ability to use free energy from some source to maintain those differences. I want to revisit the question of "what is alive?"
You will read a little about all of these. Your main assignment will be to study an adjacent pair in some more detail. The key question I want us to address for all of them is: "How and where does this replicate?" and "How does it get from one host to the other?" For the pair you study, you should be able to discuss "What's the difference between them?"
After discussing our findings, I want us to decide whether a "line" can be drawn anywhere on the list differentiating "alive" from "not alive." I will assign each of you a pair. More than one person may have a pair. For Thursday, I want to be able to discuss this as a group.
The Players
  1. Mycoplasma
  2. Rickettsia
  3. Pandoravirus
  4. The envelope virus Vaccinia
  5. Adenovirus
  6. Phage mu is a virus that infects bacteria (phage).
  7. Fertility factor or "F" factor in bacteria. This is a plasmid that cannot make virus particles that leave the cells. It is on this list for two reasons: the clever solution it takes to replication and how it gets from one host to the next.
  8. Transposon TN10. I could have picked one of many transposons for this. Here's a good general site on Transposons
  9. Retroviruses (HIV would be an example)

Below are notes of mine for general use.

  1. Mycoplasma
    1. Smallest (known) free-living organism, about 0.1 micrometer (𝜇m) in size and 0.58-1.38 million base pairs (MBP) in the genome (compare this to about 2 𝜇m 4.6 MBP for E. Coli). They are bacteria, not archaea (I was mistaken earlier). They can be detritivores (decomposers) or pathogens. There are some human diseases caused by them. They have no cell wall, but do have a membrane. They are completely capable of replicating on their own and doing all the things a living thing should do. They are cellular and would pass the current definition of life.
  1. Rickettsia
    1. These are bacteria with a cell wall, generally sensitive to antibiotics. They come in many sizes, including short rods about the same size of E. coli or thread-like structures up to 10𝜇m. They are all obligate endosymbionts or endopathogens of eukaryotes. That is, they can live and reproduce only within another cell. There hosts are as diverse as paramecium to humans. There are a couple of diseases in humans caused by them. Usually, the bacterium is carried by an arthropod and is passed to the human via a bite from a parasitic insect such as lice or ticks. Rocky-Mountain spotted fever is caused by a Rickettsia. So...it's a parasite of a parasite.
    1. However, many are perfectly stable endosymbionts, particularly in arthropods. In some species of arthropod, infection with Rickettsia causes the typical parthenogenic life cycle (e.g. Female wasps laying eggs that produce only female offspring...if you give the wasps antibiotics, they produce males...weird).
    1. The species Rickettsia prowazekii causes the disease "typhus." Based on Genome sequence, it is the closest known relative of the mitochondria.
  1. The envelope virus Vaccinia
    1. This "poxvirus" is a large DNA-containing virus with a complex protein coat and an "envelope" of membrane derived from the last cell it infected. It is oblong, about 0.27 by 0.36𝜇m. Thus, it 2-3x the size of mycoplasma, but smaller than rickettsia.Its genome is slightly less than half the size of the smallest mycoplasma, encoding approximately 200 proteins.
    1. It replicates in the cytoplasm of cells. Therefore, it has to makes its own viral RNA polymerase, which has to come into the cell already packaged in the virus. It also makes its own DNA polymerase. After it takes over the cells protein-synthesis machinery, it constructs "viral assembly factories" in the cytoplasm. Specific viral proteins are exported through the golgi to the cell membrane. The assembled virus particles, including the DNA, RNA polymerase and other proteins, are transported by Kinesin (there is a specific binding site for Kinesin on the protein coat), docks with the intracellular side of the viral membrane proteins and "buds" out through the membrane, taking the membrane with it.
  1. Adenovirus
    1. This is one of the larger non-envelope viruses. It is still pretty small at about 0.1𝜇m. It's genome is double-stranded DNA and less than 50 Kilobase Pairs (KBP). It has no replication machinery of its own. It is replicated in the nucleus using the cell's DNA polymerases and transcribed using the cell's RNA polymerases.
    1. As noted before, RNA splicing was first observed when characterizing this virus' mRNA. Two proteins encoded by the "early" genes, designate E1A and E1B bind the Rb protein and p53 respectively. You remember those two, of course. This is how the virus prevents the cell from detecting it is sick and killing itself.
  1. Phage mu is a virus that infects bacteria.
    1. It is smaller, both in the size of the virus particle and in the size of the DNA (about 37KBP).
    1. It requires bacterial DNA polymerases to replicate, and bacterial RNA polymerases.
    1. The reason it is on this list is that it links to transposons. When it enters the cell, it typically inserts itself into the host chromosome. There, it is passively replicated by the host every time the bacteria replicates (note, this is called a "provirus," and is common for human viruses also).
    1. While there, it will occasionally make additional copies of itself and place those copies around the genome of the host. The details of the mechanism may come out later.
    1. The virus detects changes to the "health" of its host. If it doesn't like the way things are going, it starts directing the synthesis of virus "capsid" proteins, makes new copies of itself and kills the cell to release hundreds of copies to the surrounding area. Thus, this virus is both a transposable element and a virus.
  1. Fertility factor or "F" factor in bacteria. This is a plasmid that cannot make virus particles that leave the cells. It is on this list for two reasons: the clever solution it takes to replication and how it gets from one host to the next.
    1. Most of the time, it is replicated by the normal DNA polymerase replication method using a fairly standard origin of replication. It encodes a protein that constructs a "pilus," which is a hollow tube made of a protein called "pilin." The pilus can create a connecting tube between the "F+" cell and an F- cell. There is some argument: The DNA may pass through this "tunnel" or the pilus may lead to the membranes of the bacteria fusing more directly.
    1. It replicates via a method called "Rolling Circle." The plasmid encodes an odd complex of proteins including a helicase that interacts with several other proteins. A "nick" (cut in one of the two strands of DNA) is made and the 5' end of the DNA is covalently connected to a tyrosine (on the OH, of course), on the protein itself. The machine then unwinds the duplex allowing the cell's pol III to replicate that strand. The old strand is transferred, single stranded, to the new cell, which then makes a copy of the second strand. Both cells are now F+, and they separate.
  1. Transposon TN10. I could have picked one of many transposons for this.
    1. This is made of two "insertion elements" called IS10 elements. All they do is cut themselves out of one location and splice themselves somewhere else. The IS elements can move just themselves or all the DNA between them...which is the rest of TN10. Doesn't seem like there is any way to increase your number and there doesn't seem to be any way to get from one cell to another...yet, there they are. The key to TN10's success is that the IS elements are flanking several genes that really help E. coli out...most notably, it encodes a gene conveying resistance to tetracycline, an antibiotic.
    1. When it "hops" from one location to another while the bacterium is replicating, sometimes the cells replication machinery helpfully "fixes" the original site, really doing the transposon's job for it. So it can end up getting duplicated.
    1. Sometimes, by dumb luck, TN10 has gotten onto an F plasmid or into a virus, which helpfully carries it to another cell. So...this thing is really a molecular parasite of a molecular parasite. The reason we see it is because we like to use tetracycline...so we killed off a lot of cells that didn't have it.
  1. LINEs and transposable introns (I'll do this one).