ReadingThinkingAndWriting

Evolution, in general, functions on a truly massive time scale. For example, between the time the first fish swam during the Cambrian period and the appearance of the first jawed fish in the Devonian approximately 100 million years had passed. And though the slow evolution of complex forms of life such as ourselves from the first combinations of amino acids into small peptides is an awe-inspiring achievement, nature took more than four billion years to make that achievement.

The ability to persist sequences of amino acids, proteins, in a form transferrable to progeny along with the fact that small changes in the sequence can produce significant changes in a protein’s function have facilitated that unimaginably long journey. For example, the FOXP2 gene has been shown to be critical in function with regard to the language abilities of humans. However, the differences between the amino acid sequences in the proteins produced in humans, other great apes, and mice is actually surprisingly small. Discarding a change that has only been observed in a family with a high rate of communication deficiencies, the only difference between the FOXP2 protein in humans and its orthologue in mice is three amino acids. In fact the chimpanzee, gorilla, and rhesus macaque FOXP2 proteins are all identical to each other and carry only on difference from the mouse and two differences from the human protein… however, the difference in the communication capabilities between greater apes, humans, and mice are obviously profound.

While the Earth provides a dynamic environment for life, the timescale of large scale changes tends to be in the millions of years. Evolution, the mechanism by which life responds to environmental changes both large and small, also operates, on average, on this scale and it is known as Deep Time. This is no wonder as evolution is constrained to act through minute random changes within the sequences of the proteins that make life happen. In general, the natural evolution of a protein involves a few mutations in the base pairs, known as few Single Nucleotide Polymorphisms (SNPs), out of the several thousand that make up the code for just a small protein. Couple this with the observed rate of at which mutation occurs during DNA replication for mitosis being 1×10-9, with the fact that most mutations have no effect on the function of the protein, and the reasons for the time scale of functional gain and the stability of the genome both become readily apparent.

Now imagine the evolution of a new protein function not over millions of years, but rather in just a few days.  A new function emerging not in response to an environmental change challenging life, but rather in response to a functional goal set by a human standing in a lab. This is not the start of a science fiction writer’s prophetic tale. This is Directed Evolution, a technique that is now commonly used to enhance existing and even create new proteins in labs all over the world.

Fundamental to genetics and evolution is the fact that genes contain the code for the sequence of amino acids that make up a single protein (in general). The reason SNPs within the code of a given protein generally have no effect on the function of that protein stems from what is termed degeneracy within the genetic code. Degeneracy is, while each amino acid is specified by a three nucleotide sequence known as a codon, more than one codon exists for the same amino acid (though no codon specifies more than one amino acid). For example, the amino acid Arginine is indicated by six different codons; CGU, CGC, CGA, CGG, AGA, AGG.

We can describe rather well the process of protein synthesis from the transcription of the code onto RNA messengers to its assembly or “translation” where ribosomes string together amino acids as specified. However, past this point, our ability to predict the structure and function of a protein from its code is just recently arising north of nil. While I stated earlier that a SNP will most often have no effect on a protein’s function, if that change results in an amino acid change within the resultant protein the effect can be significant, and is often deleterious.

This is where Directed Evolution takes center stage as it gives the ability to change the function of proteins that exist in nature, and even create totally new proteins that do not, despite this lack of understanding. The possibilities for advancement, not only in medicine, but also in industrial applications such as biofuels are endless. Endless may seem a bit of an overstatement, however, when you consider that if you made one example for every possible sequence of even a small protein 100 amino acids long, you would have made more proteins than there are atoms in the entire universe.

Fortunately, we don’t have work in those kinds of numbers since John Maynard Smith showed that functional sequence segments are actually clustered when proteins of the same length are arranged to minimize the distance between similar sequences. And this is necessarily so since, as Smith realized, evolution operates primarily on single point changes so life probably wouldn’t have evolved if this was not the case (at least not on time scales that would fit in with the age of the universe).

Philip Romero and Frances Arnold at Caltech write that though nature has been searching for optimal amino acid sequences to allow life to respond optimally to its environment for billions of years, still only an infinitesimally small fraction of those sequences has been explored. This, even considering function clustering leaves a playing field that is so large that it is hard to fathom when considering what we can achieve with new sequences.  Life could only respond with changes to an existing sequence, however, in Directed Evolution, we select the starting sequence, and instead of a change in environment defining the goal, we do. It has become a common scene in bio labs to find a researcher to take a protein and decide that instead of binding to molecule A, she wants it to bind to molecule B. Then, following natural evolution’s methods, she introduces a few pseudo-random SNPs from the original protein again and again to produce a number of sequence variants. These variants are then examined to see which moved closer to her goal; became more “fit” to perform the task. The most fit is then selected as the parent protein for the next round of variant generation and the process iterates. Amazingly, most of the time it takes only 5 iterations to find the most fit sequence and end up with a brand new protein often in just a few days. Now that’s a productivity gain.

I say pseudo-random because the SNPs may be random but they are only made in a specific range in the sequence; one that is known to code for the part of the protein that is responsible for the function that is being evolved. One of the most interesting things I’ve learned about proteins is that they are actually modular. That is there are discrete functional units, called domains or moieties, within proteins that carry out specific actions such as binding to another molecule or causing the cleavage of another protein at a specific location. Amazingly, these domains can be “inserted” into other proteins thus adding that function to a protein that did not have it as it is found in nature. The resultant protein is referred to as a Chimera and it is created by splicing the DNA sequences of interest from source genes together to create a new one. The new gene is then inserted into the DNA of a bacterium, usually E Coli., and the bacteria then creates the protein through the normal mechanism described earlier. When the bacteria have proliferated to the point that there are a sufficient number of bacteria containing the new protein, the bacteria are lysed, spilling their cytoplasm into the solution in which they live.  That solution is purified and refined until it contains only the new protein and voila, you have a vial of your very own new protein.

These practices are now common place in our biology labs all over the world. The proteins the scientists are creating have been used in many applications from bio fuels to medicine. The intersection of chimeric protein design and directed evolution may well end up being the revolution that gives us the ability to end cancer, viral and bacterial infections, and virtually any other pathogenic source of human suffering and death. The work of Dr. Carl June at the University of Pennsylvania and Dr. Rider and his team at MIT might well be heralded as the beginning of a new and improved human condition.

I would like to share with you some knowledge that very few people in the world have.  The topic is a very hearty one; Immunology, considered to be one of the most difficult in the health sciences.  However, the immune system is of critical importance not only in defeating pathogens such as bacteria and viruses, but also in clearing the body of damaged tissue and eliminating cancerous growths.  I will introduce some vocabulary for the simple reason that without it, this post would become very long, even for me.  It is said that a picture is worth a thousand words, well, in Immunology, some words are worth about the same.  The source of the information and my personal opinions presented in this post is my study of Janeway’s Immuno Biology.  I’m not a medical professional, and nothing I present in this post, or any other post I may make on a health science topic, should in any way be considered medical advice, the intent of the authors of any of the texts I reference, or an authoritative explanation.  This post is solely my understanding, theories, and interpretation of the material I’ve studied.

In a short series of posts, I will introduce an immune system process with the impressive title; Somatic Recombination, or, in this context,  V(D)J Recombination.  This process is significant for many reasons outside its critical and sole function; to build receptors.  These receptors, also called antibodies, or Immunoglobulins, are capable of recognizing bits and pieces of invading pathogens and other substances that might harm the body.  Of particular significance, the building process has potentially dangerous ends because it includes not only the cutting out and elimination of sections of an individuals DNA, but also the intentional introduction of mutations into the recombined DNA strands.  Also, the process, when viewed across many Immune System cells, is an example of selection.  It is not the same, of course, as natural selection occurring at a macroscopic level, but at the basic level, it does as it involves the survival and proliferation of those cells with receptors containing the sequence of amino acids that will best bind to a given antigen; the name given to the bits and pieces which stimulate our immune system into action.

Another interesting note about this process is that it is closely related to the process by which retroviruses insert their genetic information into the DNA of their host cells, i.e. human cells, which in turn can cause disease.  In fact, the RAG (Recombination-Activating Gene) protein which directs the process is arranged differently from other human genes suggesting that it was evolutionarily adapted into our genome making Somatic Recombination possible. 

I have often felt, during my studies that humans seem to be fabric into which is woven parts of many different molecules and organisms that probably coexisted in mutually beneficial societies; a social symbiosis.  I am of the opinion that this symbiosis is the key to the successful evolution of life rather than the necessary, but misinterpreted “survival of the fittest” notion to which evolution is so often tied.  Questioning Darwin… talk about delusions of grandeur on my part.

The Immune System, so far in my study, looks to me like a living model through which the evolution of life on Earth can be seen.  First, it was necessary for me to resist the compulsion to anthropomorphize.  The Macrophage, a staple cell of the Immune System, was so named because it was seen as a “big eater”.  I think this view is detrimental to a clear understanding and research path because we are essentially speaking of energetically favorable reactions not cells with a hankering for vittles.  When I remove this lens, I see the symbiotic relationship with mitochondria as possibly allowing the re-tasking of complex energy seeking molecules to protective capacities and the establishment of self at the cellular level.  I see that its not a matter of destroying non-self, there is actually more non-self inside us than self, it’s a matter of information dissemination, examination, and a resultant action.  To illustrate, consider that molecules called MHC which present pathogen derived peptides to T cells and elicit an immuno-protective response.  These MHC molecules, will present peptides from the individual’s own healthy proteins in the same manor if an infection is not present.  This presentation should, it would seem,  result in no action by the T cells, but considering auto-immune disorders of unknown pathogenesis, perhaps this is not always the case.  Pathogenesis is the origination and development of a disease.

This is just the very tip of a large and fascinating iceberg.  I hope my explanation of Somatic Recombination in antibody production serves well in sparking interest in a study which has saved countless lives and may yet shed light on the evolution of life  itself.