Bi 11 Organismic Biology -- Lecture #1


You have already been exposed to the study of living systems at the molecular and cellular levels. For the most part, that study emphasized features that are common to all living systems, except perhaps for the important distinction between prokaryotes and eukaryotes. In this course, the emphasis will change to a different level of organization, the level of organisms . At this level, a great deal of attention will be given to the diversity that exists at this level.
When we examine a particular kind of organism, there are three general ways in which we can attempt to understand it:
When we start to discuss evolution, it is important to keep in mind that this term is used to describe three different phenomena:
  1. Change in the genetic composition of a population of organisms. Although we often speak loosely of organisms evolving, to be more precise we should only discuss the evolution of populations of organisms. The characteristics of the population change because the individual organisms represented in it are different, because they carry and express different genetic information.
    Changes in the genetic composition of a population can occur because of mutation, gene flow from adjacent populations, random fluctuations, or natural selection . Natural selection tends to increase or maintain the adaptation of the organisms in the population to their environment.
  2. The origin of new kinds (species) of organisms, by processes usually referred to as speciation.
  3. The long-term history of life on our planet. This is sometimes referred to as phylogeny , especially when categories of organisms above the species level are discussed.

The first of these usages of the term evolution refers to phenomena that are quite well understood. It will be useful to discuss these phenomena in some detail, because it leads to a definition of the term organism. I will suggest that the term organism is best defined by the role that organisms play in the process of natural selection.

Probably the simplest organisms that we know of are monomolecular organisms consisting of only a single RNA molecule. Their ancestors were a bacterial virus called Qß.
Qß was originally isolated from a sewer in the city of Kyoto, Japan. Like all viruses, Qß is a parasite , which lives by entering and exploiting a host organism. Qß's host is Escherichia coli (E. coli ), the common intestinal bacterium of humans that has been widely used for research in microbiology and molecular biology. Viruses infecting bacteria are called bacteriophages , or often just phages.
Qß is an intracellular parasite. The actively growing and reproducing stage of its life cycle can only occur inside the E. coli cell. Qß also has an extracellular stage -- an inactive particle (sometimes called a virion ) that provides it with a means of finding and infecting new host cells. Successful infection of an E. coli cell by Qß results in lysis of the E. coli cell and release of 5000 to 10000 virus particles.
Each virus particle contains one molecule of RNA with a molecular weight of about 1.5 x 106 , or about 4500 nucleotides. Analysis of the nucleotide composition does not show that A=U and G=C, as is often found when nucleic acids are analyzed. Therefore, this is a single-stranded RNA molecule, not a paired double helix. The RNA of the virus particle is covered with a protein coat that is largely constructed from 180 molecules of a coat protein with a molecular weight of 13700 daltons.
Qß can be referred to as an RNA bacteriophage, or an RNA virus. It is a relatively simple virus particle; simpler for example than the widely studied T series of E. coli bacteriophages. The Qß virus particle is very similar in morphology to polio virus.
What kind of a parasite is Qß ? Its host, E. coli , is a typical cell that carries out DNAÆDNA replication, DNAÆRNA transcription, and RNAÆprotein translation of genetic information. It is easy to see how a DNA virus could invade such a cell and use the normal information processing machinery of the cell for its own purposes. One the other hand, an RNA virus must cause an information flow that is not part of the normal cell processes. Some RNA viruses do this by causing the synthesis of a reverse transcriptase, that causes an information flow RNAÆDNA, so that the DNA replication machinery can be used for virus multiplication. Qß does not do this, but instead causes the synthesis of a replicase -- an RNA-dependent RNA polymerase -- that can be used for virus multiplication.
The replicase is not found in the virus particle. When Qß infects an E. coli cell, the Qß RNA must be recognized by E. coli ribosomes as a messenger RNA, and used to synthesize the replicase. On the other hand, to be a good parasite, the Qß replicase must be specific, so that it replicates Qß RNA but does not replicate all of the other mRNAs in the host. Qß RNA must be enough like host mRNAs so that it can be recognized by the ribosomes as a messenger RNA, but it must be sufficiently different from the host mRNAs that the replicase can pick it out as its proper substrate. This somewhat paradoxical situation is just one of several things that make Qß a fascinating organism to study.
Detailed analysis of Qß RNA shows that it contains the message for a replicase protein of about 65,000 daltons, which requires sequence information from about 40% of the length of the Qß RNA molecule. However, the functional replicase actually has a molecular weight of about 215,000 daltons, and contains 4 different peptides. Only one of these peptides is synthesized from the Qß message. The other 3 peptides are E. coli proteins, normally found as components of E. coli ribosomes. The Qß replicase peptide must be able to recognize and bind to three other peptides found in the host, and cause them to assemble into an enzymatically active particle that carries out a reaction, RNA replication, that does not normally occur in the host cell.

One important question is: How accurate is the replicase? If it makes a mistake, and inserts an incorrect nucleotide into the RNA molecule that is being synthesized, this mistake will be a mutation, that will then be replicated by the replicase in future rounds of synthesis. The molecule will have no record of the original "correct" sequence, and no way to recover from this mistake.
Actual measurements of the mutation rate for Qß replication have been made, and it is found to be about one error for every 10,000 nucleotides incorporated. This is higher -- by several orders of magnitude -- than typical error rates for DNA replication. One of the consequences of this high error rate will be that it will be difficult to find any population of Qß RNA molecules in which all of the molecules are identical in sequence. There will be natural variation in the population. This makes it possible to carry out some interesting experiments, which were begun by Sol Spiegelman and his colleagues around 1967, originally at the University of Illinois.

The first step was the isolation of large amounts of replicase from bacteria infected with Qß. With this replicase available to work with, it is possible to examine the synthesis of Qß RNA in vitro . If you set up a test tube with a solution containing replicase, the 4 nucleotides ATP, CTP, GTP, and UTP needed for RNA synthesis, and proper salts, pH, etc. and then add a little bit of Qß RNA as a primer, it is possible to measure the synthesis of Qß RNA. The newly synthesized RNA can be taken out of the test tube and used to infect E. coli , and it will then grow up and produce normal, infective virus particles. (Some special tricks are needed to get the RNA into the E. coli cell, since it doesn't have its normal protein coat.)
Another way of describing this is to say that you have created an artifical environment for the growth and replication of this organism, Qß. Energy is provided by the nucleoside triphosphates, and they also provide all of the materials needed for the growth and reproduction of Qß RNA.

Now let's extend the experiment, and set up a series of test tubes containing this artificial environment. The first tube is inoculated with some Qß RNA, and after 20 minutes a sample is removed from the first tube and used to inoculate the second tube. 20 minutes later, the third tube is inoculated with a sample from the second tube, and so on. Speigelman's initial experiments actually used a series of 75 tubes. The growth period was reduced to 10 minutes after the first 30 tubes, and to 5 minutes after the 53rd tube.
After the 5th transfer, the RNA that was synthesized was no longer capable of initiating a successful infection, resulting in the release of infective virus particles, when it was introduced into E. coli cells.
In the 75th tube, the RNA that was synthesized had a molecular weight of only about 170,000 -- about 500 nucleotides. So it was only 1/9 as long as the original Qß RNA. The total rate of RNA synthesis (number of nucleotides incorporated into RNA per unit time) was 2.6 times the original rate in the first tube, so the rate of generation of new RNA molecules was more than 20 times the original rate.

What does this mean? When these organisms found themselves in a new environment -- the artificial environment of a test tube containing replicase -- that was different from, and much simpler than, their normal environment inside an E. coli cell, there was adaptation to the new environment. In other words, evolution occurred in this laboratory experiment. The organism found itself in a simpler environment, where it was no longer necessary to be able to make replicase or coat proteins. In this new environment, survival depends on the ability to reproduce quickly, before the next transfer. Therefore, natural selection caused the evolution of a population of organisms that were smaller and more rapidly reproducing because they had discarded the unnecessary parts of the RNA molecule. The survivors were the molecules that contained only the RNA sequence that was essential for rapid and successful interaction with the replicase to make more RNA.

It is important to note that although this is certainly a very artificial situation -- a situation
that is not found in nature -- the selection that was occurring was natural selection, and not what is called artificial selection. Artificial selection is what dog breeders (for example) do when they pick out a puppy from a litter and decide that it has the characteristics that they want to maintain in their breeding stock. That is not happening here. The experimenter is not selecting the RNA molecules. They are being selected by their ability to interact with the essential components of their environment -- specifically, their ability to interact with the replicase.

By varying the conditions of the experiment, different evolutionary outcomes can be obtained:
-- Instead of reducing the time for growth between transfers, the size of the inoculum was reduced. This led to the isolation of a strain of RNA molecules that could reproduce successfully even if only one RNA molecule was transferred to a new tube. Then it is possible to grow a clone of almost-identical RNA molecules.
-- By reducing the amount of CTP in the tubes, relative to the other NTPs, a strain was produced that reproduced rapidly but had relatively low cytidine content.
-- By including an antibiotic (ethidium bromide) that interferes with replication, and antibody-resistant strain was produced.
Each experiment with a different environment resulted in the evolution of a strain of RNA molecules specifically adapted to that environment. All had roughly the same size. Later studies discovered methods for generating even smaller successfully-replicating RNAs, with only about 200 nucleotides.

The process of evolution that is going on in this very simple environment is exactly analogous to evolution in a population of higher organisms. Each RNA molecule is playing a role analogous to that of an organism in a population of higher organisms. On this basis, each of these RNA molecules is an organism -- a monomolecular organism. They share two fundamental properties with all of the creatures that we normally consider to be organisms:
  1. In the environment to which they are adapted, they can replicate themselves. They obtain from their environment the materials and the free energy needed for this replication.
  2. They make mistakes during replication -- mutations -- and these mutations are inherited -- replicated in succeeding replications.
    These two conditions are sufficient for natural selection to occur, if any of the mutations have consequences affecting the fitness of the organisms -- their ability to survive and replicate. This doesn't necessarily mean that the population will evolve. If the organisms in the population are already highly adapted to a particular environment, natural selection will simply act to eliminate mutations that reduce fitness, so that the adapted state is maintained.

There are several fundamental units involved in these processes:
An organism is most commonly a system containing many genes and several levels of organized hardware and software used in replication. Natural selection acts at the level of the organism. It does not select particular genes and eliminate them from an individual. It acts on entire organisms, determining how much their particular collection of genes is replicated and retained in the population. Therefore, we can define an organism as the unit that plays this role in the process of evolution. It is a fundamental particle in the process of evolution -- the component of a population on which natural selection acts to determine the genetic composition of the population.

The monomolecular organisms derived from Qß are at one end of the scale of size and
complexity of living organisms. Each of these organisms weighs about 10-19 grams. At the other end of the scale we can find organisms weighing of the order of 10+7 grams -- a range in mass of about 1026. Its not surprising that there will be vast differences in the characteristics of organisms over such a wide range of sizes.

If you are still uneasy about accepting monomolecular organisms as real organisms, consider the following quotation from one of the original papers by Levisohn and Spiegelmann (1968):
"Finally, a note of caution: the synthesis of self-duplicating molecules entails the risk of introducing them as laboratory environmental contaminants. As was seen in the experiments described, one molecule can take over a reaction. This potential source of confusion has in fact been realized several times in our laboratory, beginning early in 1967 soon after we synthesized our first fast-growing mutant. The initial indication that we were being inconvenienced by our own creations was a sudden inability to prepare replicase exhibiting dependence on added template. In all instances, the difficulty was traced to contamination of a commonly employed assay reagent with a fast-growing variant. These molecules are remarkably stable under a variety of conditions. As much care as is normally employed with bacteria and viruses must be exercised to exclude these molecules as unwanted intruders in experiments."

Even if these authors are not going so far as to call their molecules organisms, they are saying that they have to be treated like organisms.

As you know, Charles Darwin is credited with first clearly showing us how natural selection is an inevitable consequence of reproduction with inherited variations, and how it could lead to evolution of populations. Nowadays, natural selection seems like a rather obvious consequence of replication and mutation. Why didn't it seem so obvious before Darwin? The usual answer is that evolution was not considered seriously before Darwin because everyone accepted religious ideas of special creation that were prevalent in the mid-nineteenth century. I'm not satisfied with this answer. One reason is that the idea of origin of species by evolution is an additional step beyond the idea of natural selection, and somewhat independent of it. It's not necessary to take that step in order to recognize natural selection. In addition, the biological literature before Darwin contains lots of evidence that other biologists had thought about ideas of gradual change or evolution of plants and animals. I think a more satisfactory answer must be based on consideration of the understanding of reproduction and inheritance that was prevalent in the nineteenth century. The older view of reproduction was that when, for example, a horse reproduced, it gave birth to a horse. Then, as a second-order effect, the horse might have some specific resemblance to its parents. This view can be traced back to the Aristotelian view of reality consisting of ideals -- with particular things being imperfect representations of reality. This view fits in well with the Creationist view of unchanging species.
The modern view of reproduction is totally different. We now easily think in terms of reproduction being an exact replication of the parental genes. Variation between parents and offspring, and any similarity to other horses, for example, are second-order effects. With the older view of reproduction, it took real insight to see it the way Darwin did and end up with the idea of natural selection. For us, looking at reproduction in an entirely different way, natural selection seems rather obvious.

Finally, this leads us to the conclusion that natural selection is not a theory that explains living systems. It is a fundamental property that cannot be dissociated from living systems.

Adaptation means nothing more than the result of natural selection. This has caused a lot of controversy. It is often argued that unless adaptation measures some tangible characteristics of
organisms, the whole idea of natural selection is just circular reasoning without any meaning. This feeling has led to attempts to equate adaptation to other properties of organisms. Perhaps the most intriguing ideas have been those based on the idea that the state of highest adaptation should be definable in thermodynamic terms. For instance, it might be a state of maximum efficiency that maintains organismic complexity with minimal consumption of free energy or negative entropy. A thermodynamic model is attractive because it admits the idea that the directiveness provided by the second law of thermodynamics can be extended to biological evolution.
None of these attempts has been really successful. We probably have to accept the fact that the idea of natural selection has no predictive value. We cannot use the fact that natural selection occurs to predict the characteristics of the organisms that will arise as a result of evolution by natural selection. The best that we can do is to look at a population, understand its ecological requirements fully, and predict which individual organisms in the population appear to be best adapted to the ecological requirements and therefore the most likely ones to survive in future populations. We may often be wrong.

A reference for further reading:

L. E. Orgel (1979) Selection in vitro . Proc. Roy. Soc. Lond. B 205: 435-442.

1995 C. J. Brokaw