CHAPTER 9 RECOMBINANT DNA AND BIOTECHNOLOGY

Genetic engineering (recombinant DNA technology) was first carried out in 1973. The first product produced this way, human insulin, was put on the market in 1982.

Recombination is the transfer of new genes into a cell. In bacteria, this occurs by transformation, conjugation, or transduction. At times this occurs naturally, but in genetic engineering selected genes can be introduced with the purpose of giving a cell or organism the ability to synthesize specific new proteins. Genes are most frequently inserted into one-celled organisms such as bacteria, since the process is much simpler.

Biotechnology refers to the industrial application of genetic engineering.

GENERAL DESCRIPTION OF RECOMBINANT DNA PROCEDURES

This is the procedure as it is most often done. Number one thing to remember: it is not as simple as this makes it sound!

1. The gene for the desired protein is obtained from a donor chromosome.

2. The donated gene is incorporated into a plasmid or the genome of a virus.

3. Plasmid-donor gene complex is carried into susceptible cell through the process of

transformation.

4. Cells successfully receiving the plasmid-donor gene complex are isolated, identified,

and cultured. These cells will have the ability to produce whatever protein the gene

normally codes for. These cells can be grown in culture to form a clone, which contains identical cells

all having the new gene.

5. Protein product is recovered and purified.

HOW DO WE GET THE GENE?

Many bacteria make enzymes that have the ability to cut a strand of DNA into pieces. These enzymes are called restriction enzymes. A particular restriction enzyme always cuts into the DNA at a particular sequence of bases. In the bacteria, these enzymes apparently are produced to attack and destroy phage DNA. In the process, the cell attaches methyl groups to some of the cytosine in its own DNA to protect it.

1. A restriction enzyme that cleaves the donor DNA in the proper place is located and mixed with the donor DNA. This will result in the desired gene being cut away from the rest of the chromosome.

2. The resulting DNA fragments are mixed with the plasmid and another enzyme, DNA ligase, which can join DNA pieces together and splice the donor DNA into the plasmid. This forms a recombinant DNA molecule.

There is a problem associated with inserting eukaryotic genes into bacteria. Remember, eukaryotic genes introns (nucleotide sequences that do not code for protein) mixed in with the exons (nucleotide sequences that do code for protein). Right in the middle of a gene may be a series of nucleotides that are not really part of the gene.

Eukaryotic cells synthesize mRNA containing both introns and exons. Then the intron sequences are deleted from mRNA by a special enzyme. Bacterial cells do not have introns, so they do not have (or need) the ability to remove introns from mRNA.

To overcome this problem, mRNA which already has the introns deleted may be used. An enzyme called reverse transcriptase can be used to construct a complementary DNA strand called cDNA with no introns. In many cases, it is cDNA that is attached to a plasmid.

GETTING THE DNA IN

1. Transformation---bacteria will need to be treated to make them competent (receptive to taking in the plasmid-donor gene complex). This is most often done as follows:

a. Bacteria that are to receive the plasmid are grown in a liquid medium.

b. Calcium chloride is added to the culture and the mixture is chilled.

c. Plasmid is added.

d. Cells are suddenly heated, which makes them receptive to taking in the plasmid.

2. Electroporation—an electrical current is used to make cells receptive.

3. Microinjection—DNA is injected through the plasma membrane using a micropipette.

IDENTIFYING CELLS WHICH HAVE TAKEN IN THE PLASMID

A simple way to do this is to use a plasmid that contains 2 genes: one for the desired protein and one making the organisms that receive it resistant to a certain antibiotic. After treatment the bacteria are grown on a medium containing this antibiotic and only the ones which received the new genes can survive.

PROBLEMS

1. Finding a restriction enzyme that will cut out exactly the portion of DNA needed can be difficult.

2. Plasmid will not enter all cells.

3. Some cells receiving the plasmid will recognize it as foreign and destroy it.

4. Extraction and purification of the product may be difficult.

APPLICATIONS OF GENETIC ENGINEERING

1. Genetically engineered products for medical therapy

a. Human insulin

b. Human growth hormone

c. Hepatitis B vaccine

d. t-PA

e. Erythropoietin

f. Interferon

g. Clotting Factor VIII

h. Others shown in Table 9.1 P. 268

2. Diagnostic tools—DNA probes for the presence of particular genes of pathogens can test for the presence of that gene (Southern blot)

3. Genetic screening---DNA probes test for the presence of specific defective genes (Cystic fibrosis, Huntington’s disease, inherited breast cancer)

4. Gene therapy---we may eventually be able to insert normal genes into cells of people with genetic defects

ETHICS

1. What if we accidentally created very dangerous pathogens?

2. Should we tamper with the genes of a person?

3. Should we screen for genetic diseases and how would we control access to this information?

4. Could new biological warfare weapons be developed?