Earlier investigations explained how nerve receptors found in humans and many other organisms can sense aspects of their environment (touching & hearing and tasting & smelling) and conditions inside their body (pain). This information is converted to electrical signals (described in pain and nerve messaging) in the form of a rapid reversal of electrical potential (a Voltage spike) across the cell membrane of neurons (nerve cells). The receptors use a chain sequence of chemical reactions in proteins embedded in the cell membrane powered by energy stored in the normal segregation between ions outside and inside the cell. This energy is initially used to generate the signal and subsequently to bolster the electrical signal at intervals as it travels along the long thin strand of the neuron called an axon over distances of up to several meters in large animals.
That signal is transferred from one neuron to one or more other cells, such as other neurons in the spinal cord or brain, to a muscle causing a contraction, or to a gland secreting an enzyme or hormone. This transfer occurs at specialized junctions called synapses. A single neuron in the brain has typically 1000 synapses where it receives signals from other neurons. The incoming signals are received almost exclusively on the neuron's dendrites, projections which extend out some distance from the bulk of the cell. The neuron then shares the signal with other brain neurons via a long thin strand called an axon, which makes synaptic connections with roughly 1000 other neurons. Thus the signal is widely shared among the billion or more neurons in a typical human brain. This information sharing provides for analysis, response, and memory.
The term synapse was derived from the Greek contraction synaptein by Charles Scott Sherrington (1857 to 1952), a British scientist who shared the 1932 Nobel Prize in Physiology or Medicine with Edgar Douglas Adrian (1889 to 1977) for their work with neurons. Sherrington and his colleagues coined the term for the then hypothetical junction from the Greek syn- meaning
together and haptein meaning
to clasp. After early experimenters such as Galvani obtained evidence that the body responds to electrical shocks, it was widely presumed that the electrical signal was passed along the nerves and jumped across the small distance between nerves just as sparks jump across small gaps between wires. In the early 1950s John Carew Eccles (1903 to 1997 Nobel photo at left ←) a student of Sherrington and Bernard Katz (1911 to 2003) developed convincing evidence that signals do not spark across synapse but rather operate by releasing packages of chemicals. Eccles shared the 1963 Nobel Prize in Physiology or Medicine.
Balloon-like membrane capsules containing signal carrying molecules called neurotransmitters lie tethered just inside the neuron's cell membrane at the synapse. When the Voltage spike mediated by a flood of ions arrives at the terminal after traveling along the long axon, Voltage-gated Calcium channels spring open permitting an influx of Calcium ions, Ca+2, into the nerve. These channels remain open only briefly. But once some Calcium ions are inside, they cause a biochemical cascade which leads to a proportional number of storage capsules moving to the cell membrane where their lipid membrane dissolves into the outer membrane. The capsules thus rupture releasing their contents of neurotransmitters out into the gap of the synapse. Randomly moving with thermal energy, some of the neurotransmitters quickly diffuse across the synapse. While the synapse gap is generally about 20 nm wide, some neurotransmitters may only need to travel 4 nm from their ruptured capsule to receptors protruding above the membrane of the receiving cell. There many of the neurotransmitters chemically bind to matching receptors. Each activated receptor in turn triggers ion channels to open in the receiving cell's membrane, allowing ions to flood across, changing the electrical potential across the lipid membrane of the receiving cell. Depending on the strength of the signal and other factors, this causes a collection of responses within the receiving cell.
When the signal traveling down the nerve axon reached the terminal, Calcium ions entered through gated channels with access through the cell membrane provided upon a rapid change in electrical potential (measured as a Voltage spike). (These Voltage-gated channels were described in the previous investigation.) There are a variety of such channels which biochemists currently distinguish by the proteins and neurotoxins which effects them. (More about that in Biochemistry 7.) Generally each channel is composed of 4 or 5 distinct protein subunits, each with six coiled α helical segments crossing the membrane with a 7th segment between segments 5 and 6 providing a gate for the pore. A positively charged amino acid, either lysine or arginine, on the 4th segment serves as the Voltage sensor which triggers gate opening.
The capsule containing the neurotransmitter is not a simple balloon structure. It's surrounding double lipid membrane typically contains a single protein pump which collects and loads the neurotransmitter into the capsule. But the membrane also contains about 180 other proteins with segments dangling out from nearly a quarter of the membrane's surface (depicted at right→ from Cell, © 2006). Its most plentiful protein (acronym SNARE for soluble N-ethyl-maleimide-sensitive factor-activating protein receptor) facilitates fusing with the neuron's outer cell membrane.
There are a variety of molecules which function as neurotransmitters: over 300 are known! These include amino acids, primarily glutamic acid (called glutamate when it is in its common ionized form), GABA (γ amino-butryic acid), aspartic acid (the aspartate ion), and glycine, small proteins (short chains of amino acids) such as vasopressin, somatostatin, neurotensin, and simple amines norepinephrine, dopamine, serotonin and acetylcholine. The two major neurotransmitters in the brain are glutamic acid (= glutamate) and GABA. Dopamine, serotonin and others are also play crucial roles in the brain. Depending on location, each neuron usually only has a small variety of receptors, and only produces one kind of neurotransmitter.
The chemistry occurring near the synapse is somewhat more complicated than described above. Some neurotransmitter molecules fail to find a receptor. And typically even those that do are eventually released back into the synapse. There are mechanisms to both deactivate neurotransmitters and others to repackage and reuse neurotransmitters. A protein chain called clathrin bind to and polymerize forming a coating around the reforming capsules, which pinches them off from the cell membrane. Once a clathrin coated capsule has separated from the cell membrane, the clathrin coats disassembles to leave the naked transport capsule. (A similar process also buds membrane segments from intracellular organelles, as in the formation of capsule containers formed by Golgi bodies.) The more detailed molecules and processes are diagrammed below:
The first knockout mouse was created by Mario R. Capecchi, Martin Evans and Oliver Smithies in 1989, for which the three scientists received the Lasker Award in 2001 and subsequently were awarded the 2007 Nobel Prize for Physiology and Medicine for introducing specific gene modifications into mice by the use of embryonic stem cells. Knockout mice have made it possible to modify specific genes in mammals and to raise offspring which carry and express the modified gene. Since genes generally govern the manufacture of proteins, this provides a mechanism to learn the function of particular proteins. Determining the role of specific genes in an individual's development, functioning and perhaps illness has revolutionized life science and provided a new route for medical therapy.
Martin Evans (b 1941 in UK, at right at Cardiff U→) identified and isolated the embryonic stem cell of the early embryo, the cell from which all cells of the adult organism are derived. He was able to extract a stem cell, maintain it in an external cell culture, modify a portion of its DNA, and reintroduced it into pseudo-pregnant foster mother mice who then developed and gave birth to offspring whose biochemistry was governed by the modified DNA. In the 1950s, Leroy Stevens at the Jackson Laboratory (Bar Harbor, Maine) discovered that mice of the 129Sv strain have a high frequency of testicular tumors which proceed to differentiate into the wide variety of cell types such as those normally needed for a developing organism. Martin Evans at the University of Cambridge obtained 129Sv mice from Stevens and used them to find a way to grown the tumor cells in a laboratory culture, independent from a mouse. On specially prepared nutrient surfaces, these stem cells continued to differentiate indefinately to all kinds of cell types such as skin, nerve, and beating cardiac muscle. In collaboration with Richard Gardner in Oxford, Evans found a way to inject these cells into blastocysts and successfully re-implanted them into foster mice. But many of these cells still contained cancerous abnormalities. So Evans used monoclonal antibodies to identify proteins imbedded in the cell membranes (see the capsule depicted above) which distinguish normal from cancerous cells. Working with embryologist Matt Kaufman, in 1980 Evans developed a way to sort out healthy embryonic stem cells from mouse embryos. In 1986 his team described their successful, efficient means for using such stem cells to producing animals with modified genes. They exploited their procedures to create and study animals with diseases found in humans. For example they installing a selected gene so that a mouse was unable to manufacture the enzyme (hypoxanthine phosphoribosyltransferase) necessary to metabolize purine, one of the common amino acids. From Evans laboratory came the first demonstration of a gene therapy to cure the deficit in cystic fibrosis in the whole animal.
Mario Capecchi and Oliver Smithies were both seeking ways of specifically altering the mammal genes, Capecchi with a view to inserting new genes into cells and Smithies in the hope of correcting genetic defects that lead to disease. The challenge is that while DNA is a large molecule, modifying the genetic code information requires reliably making specific changes in nucleotides, small groups of a few dozen atoms. Working at a scale orders of magnitude smaller that working with cells requires finding tricky ways to extend human capabilities. Virus can inject changes in the genetic code, but they typically do that at random locations and make multiple additions. To be very useful, for example to fix most genetic code errors, precision changes at specific code locations is required. Capecchi and Smithies independently discovered that they could use a natural mechanism, revealed decades earlier in bacteria by Joshua Lederberg, to introduce short sequences of modified DNA into the chromosomes of mammalian cells growing in Petri dishes. The technique allowed them to target individual genes with exquisite precision. Martin Evans provided the method for inserting such modified genes created in the Petri dish into an embryo for development into a mature animal.
DNA can exist as a twisted pair of molecules with component nucleotides which uniquely bond together by hydrogen bonds (adenine=thymine, cytosine≡guanine). But during meiosis, homologous chromosomes which share significant sequence similarity along their length may bond together even though there are sections which do not match. Occasionally such twisted homologous sections break and are repaired with their non-matching sections switched. Capecchi and Smithies found this natural mechanism is a useful means to splicing in a desired DNA modification. They produced a homologous section of DNA which contained fully matching sections to either end of the desired modification, plus a more distant section with a fatal marker. Once the desired modification was spliced into position, the other chromosome strand with the fatal marker could be destroyed. Thus the desired modification could be made in every suitable location. Mario Capecchi (b 1937 in Italy, above right at U Utah→) decided to use a fine glass pipette to inject DNA directly into the nucleus. This proved to be an efficient method which was adopted by other investigators to introduce new genes into mice. However, the transferred gene was still introduced at random into the host's DNA. Capecchi observed that copies were integrated at only one or two locations with multiple copies forming head-to-tail. Reasoned that such could only be generated by either by (1) replication or (2) homologous recombination, he demonstrated that head-to-tail combination were generated by homologous recombination, apparently using efficient enzymes responsible for normal DNA manipulation. He proposed that any cellular gene might be modified by employing these enzymes.
Oliver Smithies, (b 1925 in UK, at right at U North Carolina/Chapel Hill→) believing that homologous recombination might be used to repair mutated genes, devised a stepwise selection procedure for recovering targeted cells which successfully carried the modified genes which he reported in a 1985 issue of Nature. This process of gene modification involves first constructing a vector containing the modified DNA. This is done by sequentially adding the desired code sequence of DNA nucleotides one at a time. The vector consists of (a) two DNA regions identically matching those to either side of where the modification is desired, (b) the modified gene DNA, and (c) two additional genes to eliminate two kinds of process errors. The vector is injected into the nucleus of the embryonic stem cells with a tiny pipette. The vector binds to the homologous chromosome with the two sections which are prefect matches. This procedure then allows the natural process of recombination to occasionally (~1 in 1000) swap the modified gene for the original one. Next the two additional genes are used to assure that only the desired modified chromosome survives. In order to make sure that the vector has been inserted, a neomycin resistance gene is included. If the vector has not been inserted then the cell will die when bathed in the antibiotic, neomycin. It is also possible that the vector has just been randomly added to the chromosome. To void that possibility, a thymidine kinase gene is included to the extreme tail of the vector, outside the regions of homology. If addition has occured by the matching and recombination as intended, then the thymidine kinase gene will not get spliced into the chromosome with the middle portion of the vector, leaving the cell insensitive to gancyclovir. If instead the entire vector has been attached to the chromosome, then the cell will die when treated with gancyclovir. Thus only cells in which the DNA has been modified at the intended site by recombination will be permitted to reproduce and develop from these cells in the host mother into a new mouse. The result is a new mouse with either a particular enzyme not manufactured (thus the name knockout mouse) or a modified protein or enzyme being manufactured as a replacement.
In current practice, the modified cell is often inserted into an blastocyst stage embryo so that the offspring is chimeric, with some cells having the modified genes and others having the original genes. In that case in-breeding several generations may be necessary to achieve mice exclusively with the modified gene. For example, cystic fibrosis was chosen for study by Smithies. His researchers reproduced many features of the human disease by knocking out a cAMP-activated choride channel resulting in defective chloride transport in epithelia of airways and intestines. Oliver Smithies and Nobuyo Maeda, also focused on hypertension and atherosclerosis finding that genetic factors may account for approximately 70% of hypertension. At least 10 genes have been shown to alter blood pressure and their gene products appear to interact in complex ways. Similarly studying genes involved in various cancerous tumors has been productive in understanding cancer and developing strategies to prevent its spread.
Consider the enzyme that accomplishes homologous recombination as a well trained librarian responsible for keeping both a copy of all foreign message content and a mirrored backup. The backup, like that used for DNA, is not like a photocopy duplicate of the original but encoded in a system such as using letters a matching distance from the other end of the alphabet: A=Z, B=Y, C=X..., T=F... The code employs word-like combinations that identify where a message starts and ends so that if no message in found in a section of code, it may be found when viewing the matching copy. Actually the librarian probably can't read the foreign messages, but only knows enough of the nature of the code to recognize this mirror like matching. Since radiation and corrosive chemicals occasionally cause tears, random repairs and other other damage, the librarian's responsibility is to periodically compare the normally matching code and backup copy for consistency. If a break, omission, or unmatched section if found, the solution is to incorporate the extra content in the primary copy. While the librarian can't read the code to determine if the content contains a message, the assumption is that if there is a contained message it will be recaptured and eventually also backed up. If no start-end is found in the content, then perhaps the eventual matching backup might contain a message with appropriate start-end signals. But in any case, incorporating any discovered additional content is useful for preserving any retrievable message following content damage. Thus the enzyme that tends the DNA code incorporates any discovered additions into the primary DNA library of messages.
To better understand this process, you are assigned the responsibilities of DNA librarian to compare the following two strands of code, first finding the sections which are matching, then identify any potential message carrying content that exists only on one strand in between the matched sections:An actual vector is needed here, or at least an abbreviated one. Any suggestions?
Communicating technical information such as observations and findings is a skill used by scientists but useful for most others. If you need course credit, use your observations in your journal to construct a formal report.