Simulation of Gene Splicing
Judith Averbeck, SND
1994 Woodrow Wilson Collection
This exercise may be used as a prelude to a "wet" lab or as a substitute for such a lab. It correlates well with colony transformation labs. It is recommended for students who have difficulty with the abstractions that genetic engineering involves. Students need to have some knowledge of bacterial morphology, DNA structure, restriction enzymes, etc. before doing this exercise.
The teacher may want to ask the students to do the background reading and its accompanying questions before the lab period.
- Paper of two colors onto which the DNA sequences have been copied
- Transparent tape
Human Growth Hormone
Terry's biology class had been studying human inheritance and were now discussing genetic defects. When Terry heard about dwarfs and midgets, he began to think about his sister, Julie, who had always been shorter than normal for her age. He remembered the concern of his parents when Julie was younger, especially before some sort of "treatment" that accelerated her growth had begun. When the teacher assigned oral reports to be given on various human defects, Terry resolved to do one on "whatever Julie has." The teacher accepted this, but cautioned that Julie's condition might not be genetic.
That night, Terry talked to his parents. They confirmed that Julie's growth problems were indeed inherited and suggested that Terry go with his mother on her next visit with Julie to the doctor. He knew that Julie went every week to get shots and that these seemed to be helping because now the difference in height between her and her peers was not nearly so great now as it had been when she was a little kid. In fact, Julie seemed pretty normal for a 10-year-old, Terry thought. Sure, she was a girl and sometimes a pest. But, overall, they got along and even shared some interests like soccer and music.
The next Monday, while Julie was getting weighed and measured, Terry and his Mom sat down with Dr. Brown who was an endocrinologist specializing in children's problems. Terry explained about his need for information for his report and the doctor seemed willing to spend a little time helping him out. She began by saying that a person's height depends on many factors. Among these are genes, hormones and nutrition. "Julie's difficulties," she continued, "were diagnosed as being mainly the result of growth hormone deficiency. This hormone is normally produced by the pituitary gland in the brain, but in Julie's case there didn't seem to be enough of it. As a result, when her pediatrician noticed on Julie's growth chart that she was lagging behind the average growth rate, he did some special tests and concluded that insufficient quantity of hormone was the culprit. Fortunately, we were able to treat Julie by injections of the hormone that have produced close-to-normal growth."
After leaving the doctor's, Terry decided to go the library and search for further information. There he read that human growth hormone is really a protein consisting of 191 amino acids. Some people who lack the normal amount of this hormone inherit the deficiency from their parents in a recessive manner. Terry felt very intelligent at this point because he knew that meant that he had had a one in four chance of inheriting the condition from his carrier parents.
Terry also learned some new facts. For many years, there was no treatment for those lacking sufficient hormone. In the l950's, it was found that hormone from the pituitaries of dead people could be used as a treatment. However, not enough people donated their glands to supply hormone for all those who needed it. Even more sadly, some of the pituitaries used for this purpose contained a deadly virus. Unknowingly, doctors had injected the virus into some children along with the hormone and they had died.
Then came genetic engineering. In the l980's, scientists figured out a way to cause a bacterium to produce human growth hormone. This was an important breakthrough because the technique provided large quantities of safe hormone at a reasonable cost. Terry thought this new way to produce a human molecule sounded pretty neat. He was grateful that such a discovery allowed Julie access to a safe, reliable source of what her body could not itself produce and assured her fairly normal growth for the rest of her years.
- Where does the growth hormone in your body originate?
- How many nucleotides of DNA code for the production of human growth hormone?
- What is the mode of inheritance of human growth hormone deficiency?
- Name some advantages to using genetically engineered growth hormone over other sources of it.
- How does one know if one has growth hormone deficiency?
Setting the Stage
Before we can understand how, through genetic engineering, bacteria can provide human growth hormone, we need to recall some things we know about bacteria and their DNA.
E. Coli bacterium
Enlarged Plasmid (with gene for Ampicillin resistance enclosed in brackets)
NOTE WELL: throughout this exercise, the number of nucleotides used to represent genes, plasmids, etc. is far, far fewer than the actual number. This is done for the sake of convenience.
In the drawing above is an E. coli bacterium and the plasmids it contains. One of these is the plasmid we will use to carry the human growth hormone gene into a new bacterium. It is shown enlarged. Note that it carries the gene for ampicillin resistance.
- In the picture of the E. coli, label the plasmids and the large "loop" near the middle.
- What are plasmids?
- What bonds in the DNA of the enlarged plasmid are not shown?
- Note that the enlarged plasmid contains a gene for ampicillin resistance. Normally, the antibiotic ampicillin will kill E. coli bacteria. If this gene for resistance is present, however, it will permit the bacterium containing it to "resist" the power of the ampicillin and continue to live in its presence. (Actually the gene enables the bacterium to synthesize a protein enzyme that inactivates ampicillin before it has a chance to kill the bacterium.)
If the E. coli shown above were grown on agar containing ampicillin, what would happen to the bacteria? Would they be able to grow and reproduce or not? What about E. coli from which the plasmid shown had been removed? Explain.
Splicing the Growth Hormone Gene into a Plasmid
- The plasmid shown above in "flat" form is represented on a separate sheet of paper you have been given. Cut out the strip of DNA and tape its two ends together. You now have the same plasmid as shown above except in 3-D form. This is the bacterial DNA into which you will insert the human DNA (gene) that codes for growth hormone.
- A section of human DNA is shown below. This section contains the gene for human growth hormone.
C C C T G T A T A A G C T T A T G G C T A C A G G C T C C C G G A C G A A G C T T A
G G G A C A T A T T C G A A T A C C G A T G T C C G A G G G C C T G C T T C G A A T
The m-RNA involved in the synthesis of human growth hormone was isolated and found to be:
G A A U A C C G A U G U C C G A G G G C C U G C
Study the human DNA and locate the portion that is the gene for growth hormone. Underline or highlight this section.
- Now that the plasmid DNA and the human gene DNA have been determined, the next step is to cut each one as a prelude to combining them. The growth hormone gene must be cut out of the human DNA and the plasmid (bacterial DNA) must be cut open before the human gene can be inserted. The scissors for doing the cuts are restriction enzymes. Below is a short list of a few of the enzymes available. Study the list carefully and select the enzyme that would be appropriate for cutting out the growth hormone gene from the human DNA and for cutting the plasmid open. If some "unwanted" bases are included in the human piece of DNA, no harm done.
- Once you have selected your enzyme, you have identified the sites at which the two DNA's will be cut. Using your 3-D paper plasmid and the human DNA found on the separate sheet, mark IN PENCIL the sites at which the enzyme will cut. When you are sure the marks are in the correct positions, cut the ends with scissors. This will produce uneven ends which have unpaired bases. Study these ends and decide how the human DNA can be joined to that of the bacterial plasmid. Tape the pieces together. You now have "recombinant DNA."
- Which enzyme is the correct one to use in this exercise? Explain why in detail.
- In a real gene splicing, what kind of "tape" would be used?
- What sticky ends have you made on the human DNA containing the growth hormone gene? What sticky ends have you made on the bacterial DNA (plasmid)? Compare the two. What do you observe?
- Once the recombinant DNA you just constructed was in existence, the next step would be to insert it into a new bacterium. It would be very important to know whether any given bacterium had really taken in the recombinant plasmid and so acquired the ability to make growth hormone. What characteristic, in addition to the ability to synthesize human growth hormone, should any bacterium that took up the recombinant plasmid possess? (Hint: look back at the "flat" representation of the bacterial plasmid)? Explain why.
- How could you test to see if the bacterium had indeed taken in the plasmid?
- What exactly is recombinant DNA?
Answers to Background Questions:
- In the pituitary gland
- 573 (3 x 191)
- Autosomal recessive
- No danger of viral infection; larger quantities available; cheaper; virtually unlimited supply.
- Growth is stunted. Becomes apparent in childhood if height is compared to average height of peers.
Answers to Setting the Stage Questions:
- The large loop near the center of the bacterium is its circular chromosome. The smaller circles are plasmids.
- Plasmids are small loops of DNA found in the cytoplasm of bacteria.
- Hydrogen bonds between bases are not shown.
- If the E. coli shown were grown on ampicillin-containing agar, it would grow and reproduce because it contains a plasmid carrying the gene for ampicillin resistance. If the plasmid were removed, the bacterium would be killed by the ampicillin.
Answers to Discussion Questions:
The gene for human growth hormone that should be highlighted is:
C T T A T G G C T A C A G G C T C C C G G A C G
- HindIII is the correct enzyme to use because it has cutting sites on both sides of the growth hormone gene and thus will cut it out intact. In addition, it has a cutting site on the plasmid and will thus "open" it for human gene insertion.
- In real gene splicing, ligases are used to reattach the cut ends of DNA.
- The sticky ends produced (A G C T T and T T C G A) are complementary to one another and should bind together.
- The bacterium should possess ampicillin resistance since the plasmid we are using contains that gene.
- You could attempt to grow the bacterium on agar containing ampicillin. If it had taken in the recombinant plasmid, it would grow successfully and reproduce. If not, it would not survive.
- Recombinant DNA is DNA from two or more sources that has been "spliced" together.
(This exercise is based on a lesson from: BSCS. Advances in Genetic Technology. l989. Heath.)
TO BE COPIED FOR EACH STUDENT ON PAPER
Human DNA (Containing Gene for Growth Hormone)
C C C T G T A T A A G C T T A T G G C T A C A G G C T C C CG G A C G A A G C T T A
TO BE COPIED FOR EACH STUDENT ON PAPER OF A DIFFERENT COLOR
G G G A C A T A T T C G A A T A C C G A T GT C C G A G G GC C T G C T T C G A A T
Plasmid DNA (Bacterial DNA)
G G A T C C T G A C A C C G G A A C G T C A A G C T T C C C
C C T A G G A C T G T G G C C T T G C A G T T C G A A G G G