CLONING AND b-GALACTOSIDASE

The idea of cloning mammals was reawakened in the late winter of 1997 when Dr. Ian Wilmut created a Finn Dorset lamb that he named Dolly. Dolly had been cloned from the DNA of mammary cells of a Finn Dorset ewe. It took 277 tries before he achieved success. 277 eggs were used but only 29 embryos survived longer than six months. Except for Dolly, all embryos were dead. What was truly exciting about this achievement was that Dr. Wilmut was able to take a cell that had been differentiated (DNA folds up in the nucleus making vast stretches of it inaccessible to turn on at the wrong time and in the wrong tissue.) and turn it into an undifferentiated cell. [5-8 Content Standard E- Understandings about science and technology]

In a New York Times article (July 23, 1998), Dr. Ryuzo Yanagimachi of the University of Hawaii used the same approach as Dr. Wilmut to clone 50 mice. The only exception is that Dr. Wilmut used electricity to fuse the nucleus and egg cell together and Dr. Yanagimachi, once he inserted the nucleus into the egg mechanically, used chemical methods to start the reaction. If mice and sheep can be cloned, why not humans? Richard McCormick, a Jesuit priest and professor of Christian Ethics at the University of Notre Dame finds cloning of humans morally unacceptable. Most religious groups would probably concur, but something might be said of cloning for health benefits. A human clone can serve as a source of bone marrow (or a paired organ) if the donor should become ill. Or even better, an organ by itself can serve as a replacement if the donor's organ should become defective. [5-8 Content Standard F- Science and technology in society] This process is called tissue engineering. In an article in Discover magazine (May 1998) it is predicted that tissue engineering will eventually be the source of all cloned hearts, kidneys and most everything else except the brain.

Most students in an intermediate school are well aware of these findings from the various news media. It would be beneficial for them to gain first hand empirical knowledge.

A clone may be defined as a group of genetically identical cells descended from a single common ancestor. Most clones are from asexual reproduction, a process in which only one parent forms the new individual. A plant formed from vegetative propagation, a form of asexual reproduction, will be a clone. Many seedless varieties of flowering plants such as oranges and grapes are reproduced only through vegetative propagation provided that spontaneous mutations do not take place. Single-celled organisms such as bacteria and yeast usually reproduce asexually. [5-8 Content Standard C- Reproduction and heredity]

A clone may also be defined as a replica of a DNA sequence, such as a gene, produced by genetic engineering. Genetic engineering entails eliminating or introducing specific genes into the nucleus. Plasmids found in certain kinds of bacteria and yeast can be used to transfer a gene by this process. Plasmids are circular pieces of DNA that exist separately from the main chromosomes. They contain very few genes and only those plasmids that confer some selective advantage will be maintained in a population. One advantage, antibiotic resistance, is beneficial when a bacterium has to compete against another microorganism that secretes antibiotics. By cloning such a plasmid many identical copies will be produced.

PUC is a family of plasmids that have an ampicillin resistance gene and more importantly a lacZ gene. A functional lacZ gene will produce the protein b - galactosidase. Bacterial colonies in which b - galactosidase is produced, will form blue colonies in the presence of the substrate 5 - bromo - 4 - chloro - 3 - indolyl - b - D - galactoside or as it is more commonly referred to, X-gal. A mutant that has a nonfunctional lacZ gene will form white colonies. This simple color test to determine whether bacterial cells that have been cloned with a functional lacZ gene will lead to our laboratory investigation. [Content Standard Unifying Concepts- Change, constancy, and measurement]

Some of the techniques developed in Dr. Lieberman=s lab will be incorporated into this year=s laboratory exercise. Namely, the identification of E.coli cells that contain a pUC vector (plasmid.) This vector carries a lacZ gene and a gene for ampicillin resistance. The lacZ gene produces the protein b - galactosidase. Bacterial colonies that have b - galactosidase will appear blue on agar containing X-gal.

A portion of last year=s action plan will be incorporated into the 1998-1999 school year. The analysis of Vit. C in different orange flavored drinks (for inquiry skills) and the assay of proteins in plant products. This assay makes use of coomassie blue and BSA (bovine serum albumin.) Both activities will introduce students to the joy of biochemistry. At the end of this packet, the protein assay experiment will be included.

PROBLEM: How can we determine if E coli cells contain b - galactosidase?

MATERIALS: Petri dish containing nutrient agar with X-gal, petri dish containing nutrient agar without X-gal, 2 - 1mL pipettes, rubber bulb, E coli cultures, alcohol lamp, inoculating loop, marking pencil, ampicillin disks. [Teaching Standard D- Make accessible science tools]

1. Obtain a petri dish with X-gal and use your marking pencil to draw a line down the middle of plate. (NOTE: Draw on bottom of dish that contains the agar.)

2. Write the number 1 on the left side and 2 on the right side. Again write on bottom of dish.

3. Turn the plate over and put the initials of your group and today's date. (NOTE: Print around the circumference.)

5. Attach a rubber bulb to a 1mL pipette and use sterile techniques to obtain 100 mL of E coli suspension 1. (100 mL = .1 mL)

6. Lift cover and inoculate the bacteria on the petri dish labeled one. (Near center)

10. Repeat steps 4 - 9 for bacterial suspension 2. (NOTE: Use right side of petri dish.)

12. Place plate in incubator set for 37ºC and check daily for growth of colonies.

DATE	SIDE 1 COLOR---------COLONIES	SIDE 2 COLOR---------COLONIES

2. How do we know if it is present? [5-8 Content Standard A- Use tools to gather and analyze data]

5. Most plasmids confer antibiotic resistance. Use the steps of the scientific method ( problem, hypothesis, materials, procedure, observations and conclusions) to determine whether one or both cultures show resistance to ampicillin.

The history of cloning plants and animals will be researched by the entire class. [5-8 Content Standard G- History of science] Each student will submit a paper for evaluation. Selective students, hopefully volunteers, will also research the positive and negative aspects of human cloning. This information will be used for a debate entitled, "Should human cloning be allowed?" [Teaching Standard B- Orchestrate scientific discourse]

Kolata, G. In Big Advance, Cloning Creates Dozens of Mice. The New York Times: 7/23/98

Micklos, D.A., and Freyer, G.A. DNA Science. New York: Cold Spring Harbor Laboratory Press. North Carolina: Carolina Biological Supply Company, 1990.

Sambrook, J., Fritsch, E.F., and Maniatus, T. Molecular Cloning (A Laboratory Manual). New York: Cold Spring Harbor Laboratory Press. 1989.

The American Heritage College Dictionary, Third Edition. Clone. New York: Houghton Mifflin Company. 1997.