Genetics and Human Life - Part 2

You will notice in this past several lectures that they build on a lot of the concepts you will have learned in earlier weeks.

The biology I talk about today may become part of your future. At some time you may sit on a jury which has to decide guilt or innocence based on the evidence presented from biotechnology. It is likely that we all will be asked our opinion regarding regulation of biotechnology. I owe it to you to tell you about the "cutting edge" of biotechnology, and you owe it to yourself and possibly even your future children to learn as much about it as you can.

Biotechnology is heavily technique-driven. You will need to understand at least four biotechnology techniques:(1) cutting with restriction enzymes (REs), (2) gel electrophoresis, (3) cloning within bacteria, and (4) polymerase chain reaction (PCR).

Then, I want to look at two applications of these techniques: (1) DNA fingerprinting and (2) recombinant DNA. Finally, I will take a quick look at what one can rationally expect the future to hold with biotechnology and why you should not expect wings on elephants.

When one speaks of genetic engineering, it may sound like researchers manipulate genes with sterile tools such as microscalpels and needles. In fact, as you will see, most of the tools used are biochemicals derived in large part from bacteria.

In an earlier module, you learned that the message encoded in DNA is contained in the linear sequence of its base pairs. The first problem then is how to remove a piece of this sequence to study it. For "microscissors" one turns to bacteria.

You will recall that bacteriophage is a name given to a special kind of virus which attacks bacteria. Bacterial defenses against such attack include a number of enzymes (restriction enzymes) produced by the bacterium that recognize foreign DNA and snips it apart. Several hundred different restriction enzymes (REs) have been isolated from various kinds of bacteria. Each makes its snip or cut only at sites having a short, specific nucleotide sequence.

So one has molecular scissors that will snip or cut DNA at specifically chosen nucleotide sequences. Many of these cut only at unusual repeating sequences and cut unevenly, so both ends of a DNA fragment end up with a short, single-stranded tail that is "sticky." A diagram illustrating how one of these enzymes cuts is provided for you.

The sequences which are recognized by these DNA-cutting enzymes are palindromic. Palindromes are words which can be flipped backwards and still spell the same thing. Take a look at the bases in the diagram available above. Notice the GAATTC in the one DNA strand and the CTTAAG in the other strand.

Restriction enzymes produce DNA fragments. If one chooses the proper restriction enzymes and has available appropriate "probes" one can ultimately "pick" out a desired gene. The complicated story about how one kind of probe is made is provided for you here.

Gel electrophoresis is a technique that separates the various DNA fragments and allows eventual visualization of them. How does this work? Read on.

In a gel electrophoresis apparatus, a gel is cast in a glass tube or as a slab between glass or plastic plates. For the separation of nucleic acids, the gel is usually made of agarose, a complex carbohydrate obtained from seaweed.

The gel is suspended so that its ends are in contact with separate buffered salt solutions. The salts that are used conduct electricity. One buffered solution is connected to the positive electric cable; the other to the negative electric cable. Current then flows from one solution through the gel to the other solution. Any molecules (DNA fragments, for instance) added to the gel tend to migrate toward the positive or negative end of the gel, depending on their molecular charge.

The gel itself forms a mesh of a particular size. When the electric current is held constant, molecular movement depends not only on charge but also on the size and shape of the molecule.

The fastest moving DNA molecules are the smallest ones.

The DNA fragments separated in this manner are still invisible to the naked eye. They are often visualized after staining with ethidium bromide which fluoresces under ultraviolet light.

The DNA fragments are called restriction fragment length polymorphisms (or RFLPs). Use of RFLPs led to isolation of the genes for Huntington's Chorea and cystic fibrosis in the human genome.

One can use this technique to find the actual sequence of nucleotide pairs on a particular DNA fragment.

You have all read about the human genome project. At present, we have the exact nucleotide sequences for several pieces of the human genome. But just think about it- the human DNA base pair sequence is 100 times the number of strings of single letter characters in an 18 volume World Book Encyclopedia set. And finding the base pair sequences is a complex process, so we do not expect the sequencing to be finished any time soon. And when it is finished, when a single human being's genome is completely sequenced, we will have sequenced just one human's genome.

We probably have an average of 0.03% diversity among humans. Thus, in the 3 billion base pair sequences, the chart of the "average" human is likely to be different from yours and mine by at least 1 million base pairs.

There is a fair amount of controversy over the granting of patents on human gene sequences. A fair bit of the genomic sequencing will have been done by private, for profit corporations. They will make a profit with this information. What do you think of patenting the human genome?

The next set of manipulation techniques involves DNA amplification. That means making lots of copies of the particular gene fragment one has isolated. The two most common ways to do this are bacterial cloning and the polymerase chain reaction (PCR).

Bacterial cloning often involves an old friend, E. coli.

As a prokaryote, E. coli has no nucleus so its single circular chromosome is free in the cytoplasm. Earlier, you learned that bacteria can actually incorporate alien DNA that they absorb from dead bacteria (the Griffith and Avery experiments). If one inserts an isolated gene sequence in a bacterial plasmid, a tiny circular strand of DNA within the bacterium, it will be replicated each time the cell divides. A diagram of how this might look is provided.

Another way to amplify selected DNA is to insert the DNA sequence into the bacteriophage, Lambda, which integrates its DNA directly into the bacterial chromosome when it enters the cell.

Both the Lambda Phage DNA and plasmid DNA can be cut with restriction enzymes so that one can add a gene sequence to them.

We call these things good vectors for getting DNA sequences into bacteria. A diagram showing how a gene fragment cut by the restriction enzyme Eco RI is added to a plasmid cut with the same enzyme. Assume that we have snipped out the DNA sequence for human insulin from a human cell, and inserted it into a plasmid which has been placed within E. coli. As the bacteria reproduce in the beaker, each daughter cell gets a copy of the introduced plasmid. You can review the complicated story about how the DNA sequence for human insulin could be identified and used.

Bacteria may take hours to replicate so it may take days to get all the clones one wants for an experiment. A newer technique called polymerase chain reaction (PCR) can accomplish in just minutes what takes a bacterium hours to do.

Earlier, you learned that when one heats water one breaks hydrogen bonds. The same water, when cooled, will re-form hydrogen bonds.

Heat can also break hydrogen bonds in proteins. In general, this process is not reversible. When you cool a fried egg, the egg white does not revert to a clear liquid. Nucleic acids are different from proteins, however, and one can renature them after breaking their hydrogen bonds.

Here is how the polymerase chain reaction is done. First one denatures DNA with heat. The double helix becomes two separate strands.

Next, one renatures (cools) the DNA in the presence of appropriate nucleotides and chosen primers. Primers are very short single DNA strands with known sequences which will attach to the single stranded DNA bases and begin the third step, replication.

After a few minutes each single stranded fragment has replicated (made a new DNA strand and returned to double helix state). The reaction mixture is heated up again to begin the next cycle of replication. This is done over and over again. You can see how quickly large numbers of fragments are synthesized by referring to the table below.

DNA fingerprinting can utilize RFLPs, PCR or both. Minute amounts of DNA left behind in a single follicle cell attached to a pulled hair, a drop of semen or a drop of blood can be amplified with PCR to give a reading of RFLPs which can narrow the range of suspects.

DNA fingerprinting is also controversal these days. Should the government DNA fingerprint its citizens? DNA fingerprints contain genetic information. Is the government a safe repository for this sensitive information?

The use of DNA fingerprinting and blood residue may be the first thing you think of after all the publicity DNA fingerprinting received in the O. J. Simpson murder trial.

Think carefully about this for a moment. Will we get DNA from the red blood cells which give blood its characteristic color? No. Mature, circulating mammalian red blood cells have no nuclei and thus no DNA. So the white blood cells are what one looks for in blood residue.

You might have the impression that DNA types from these kinds of analyses are as individual as true fingerprints. That has yet to be demonstrated. If the DNA does not match, it can rule out the suspect as the source of the hair, blood,or semen. If the DNA does match, and the laboratory tells us that the sample source is found in only 1/10,000 of the population, that does not say that this particular suspect is the source of the DNA in question. It only says he or she could be the source of the DNA.

Moreover, the population figures for DNA types come mostly from the laboratories that designed the tests and they have used extremely small samples upon which to run these $1,000 tests. Statistical validity depends on large sample sets; their statistics could be very wrong. At this point DNA fingerprinting should be used only as corroborative evidence in cases where there is other physical evidence. But it is pretty good.

Statistically small sample sets were found to be inadequate in a court case in the UK recently. A person convicted on the basis of DNA evidence insisted upon additional testing and when this was done with a larger sample set, he was found to be not the guilty party. We have to be very careful here.

Recombinant DNA, placing a piece of DNA from one organism into another, creates what we term a chimera. In Greek mythology the Chimaera was a monster with the head of a lion, the body of a goat and the tail of a serpent. Our modern chimeras are simply organisms which have some part of their DNA introduced from another organism.

In order to recombine DNA you need to choose a target tissue or organism, a vector, a marker and a promoter and an isolated gene sequence. The easiest sort of target organism to use is a bacterium, just as we saw in cloning. The two vectors we discussed, Lambda phages and plasmids, are excellent vectors for bacteria.

One also needs to incorporate a genetic marker, so that we will be able to know when recombination has occurred. A commonly used marker is antibiotic resistance. Other markers include digestive enzymes and luciferase.

If you marked with antibiotic resistance, you then culture your bacterium in a petri dish filled with the antibiotic in question. The bacteria which survive will be those which incorporated the gene you spliced in.

If your marker was a digestive enzyme such as beta-galactosidase, you add lactose to your culture medium and only those bacteria which secrete this enzyme will be able to digest it and grow.

Luciferase, is an enzyme naturally synthesized by fireflies which combines with ATP and luciferin to make these little beetles glow at night. If you marked with luciferase, your successful recombinants will then be able to glow.

Finally, we need a promoter for the chosen gene sequence. You learned earlier how RNA polymerase attaches to the promoter site and begins gene transcription. Without a promoter there will be no way to translate the gene into a protein product.

One of the first chimeras produced and a very important medical one, was the placing of a gene for human-type insulin into E. coli.E. coli was a good target for the gene since we know a lot about its genome. Moreover, because prokaryotes don't need to send their mRNA out of a nucleus (they haven't any), ribosomes and tRNA can attach to nascent mRNA and begin translation while still being transcribed. They can churn out proteins much faster than eukaryotic cells because of this.

But getting the eukaryotic insulin gene into this prokaryote involved one more step. You know that eukaryotes process their mRNA by snipping out large sections to "mature" it before it is ready to be translated into protein. Prokaryotes do not process their mRNA so we must snip out all the DNA sequences that will code for mRNA that would otherwise be snipped out before inserting it into a prokaryotic plasmid.

The easiest way to remove this DNA from the insulin gene was to start with "mature" mRNA. You remember this is synthesized by pairing RNA bases to the sense strand of DNA. What must now be done is reverse transcription, much like what you read about earlier- make a strand of DNA from mRNA.

Although prokaryotes such as E. coli are an easy target organism to work with, often we need to use eukaryotic target tissue. In plants, this involves removing the cell wall. And the vector will be bombardment of the naked cell or protoplast with microprojectiles (minute silver bullets coated with DNA). We can use a cell culture for this and then grow the culture, adding plant hormones, until it becomes a seedling. Plants are open systems so our recombinant DNA will have a chance to be in pollen and egg cells, and get into future generations.

Animals are not open systems so we must choose between a somatic cell and a germ line cell. Drosophila species, which are insects commonly called fruit flies, are among the best studied organisms in genetics. They may also have the widest range of possible vectors in the animal world.

The Drosophila genome contains a number of transposons, genes which literally jump from chromosome to chromosome. Drosophila are also infected by baculoviruses (which affect only insects and not people) which can be made to carry extra genes.

A few years ago, Science published an article on the eyeless gene of Drosophila. The promoter (opposite of eyeless) was placed in other parts of the genome and the result was flies with eyes in the back of their head (in addition to the two normal eyes). Many of the flies also had eyes on their legs and backs.

Some important results of biotechnology include developing bacterial factories for human gene products from insulin to interferon. Chimeric bacteria can function as chemical factories, more accurate and efficient than any human-run laboratory.

Transgenic mice often can function as a disease model for humans, with a large role in discovering a cure for a particular disease.

Exciting research is going on now with transgenic pigs whose organs produce the human enzyme which halts our immune response. Close to us in size, but much faster-growing, they could make ideal organ donors.

The future will hold even more recombinant E. coli making gene products of medical importance. More research into eukaryotic gene function (such as eyeless) will be done on Drosophila. Drosophila may replace mice in a lot of drug testing but we will still need mice, transgenic mice, to study the effects of new drugs on people. There is bound to be a lot more gene therapy and more discoveries of cancer causes and cures as spinoffs of basic research.

But there are many things we will not see. It is doubtful that there will be a real Jurassic Park for a long time, if ever. Remember the mosquito in the amber? That was a male mosquito and only female mosquitos suck blood. There was no blood in the gut of the mosquito they showed us on screen.

Also did you wonder why they filled in missing dinosaur genes with amphibian genes and not genes from a much closer relative, the crocodile? Of course, amphibian genes assist the plot and allow the all female populations to reproduce, but the geneticists working on such a project would never manage to get such sloppy work published. In fact, the creations, according to their shoddy construction, should have looked more like frogosaurs!

And we will not be tailoring an organism which defies the laws of physics such as a winged elephant. First, what kind of wings? Bird wings are derived from the first pair of legs (or arms) in vertebrate ancestors. So unless we can find a six legged elephant, we will have to turn the front legs into wings. But our winged elephant won't fly as long as he has elephant bones. Bird bones are also hollow to make the animal lighter for flight. So, we would also have to move the gene(s) for hollow bones to the elephant. Could such a skeleton support such a massive animal?

Our winged elephant cannot walk, having lost his front legs, but he still can't fly. The poor creature may not even be able to lie down or sit.

So back to the drawing board; maybe the fantasy creature, Dumbo, would be a better idea. We will be working with existing tissues, the ears. We would have to enlarge and strengthen them, and when they get large enough our little elephant might be able to get airborne. But he would lack any maneuvering system so once he is airborne, he would have a control problem. Putting functional wings on an elephant would not be a very easy task.

That's it for the genetics material. You might wish to go back to the first genetics module.

You may take a quiz on the material in this module. No record of the quiz is made. You decide after the quiz if you really know this material.

Or you can return to the Syllabus Page.