TRANSFORMATION

By 1926 the quest to determine the mechanism for genetic inheritance had reached the molecular level. Previous discoveries by Gregor Mendel, Walter Sutton, Thomas Hunt Morgan, and numerous other scientists had narrowed the search to the chromosomes located in the nucleus of most cells. But the question of what molecule was actually the genetic material had not been answered.

In 1928 Frederick Griffith, in a series of experiments with Diplococcus pneumonia (bacterium responsible for pneumonia), witnessed a miraculous transformation. During the course of his experiment, a living organism (bacteria) had changed in physical form.

The pneumococcus bacterium occurs naturally in two forms with distinctively different characteristics. The virulent (S-strain) form has a smooth polysaccharide capsule that is essential for infection. The nonvirulent (R-strain) lacks the polysaccharide capsule, giving it a rough appearance. Mice injected with S-strain of the pneumococcus bacteria die from pneumonic infection within a few days, while mice injected with the R-strain bacteria continue to live. Injection with heat-killed S-strain bacteria also results in the mice surviving.

Griffith was surprised to find in his experiments that mice injected with a mixture of heat-killed S-strain and live but nonvirulent R-strain produced lethal results. In fact, Griffith discovered living forms of the S-strain bacteria in the infected mice !

He hypothesize that the R-strain bacteria had somehow been transformed by the heat-killed S-strain bacteria. Some "transforming principle", transferred from the heat-killed S-strain, had enabled the R-strain to synthesize a smooth polysaccharide coat and become virulent.

Oswald Avery, Colin McCleod, and Maclyn McCarty (1934-1944) at the Rockefeller Institute, building on Griffith's work, showed that only DNA could cause the transformation. They isolated a cell-free extract from the S-strain bacteria and were able to transform living R-strain into a culture containing both S-strain and R-strain cells. The purified extract contained Griffith's "transforming principle". Through biochemical testing, they showed it to be deoxyribonucleic acid (DNA).

Classroom Transformation

The transformation witnessed by Griffith is a random and rare event. Today it is possible to reproduce Griffith's transformation in the classroom in a more controlled and reliable process. Requirements to complete the process include:

1. A host bacterium for gene insertion.

2. A plasmid, self-replicating vector, to carry the

foreign gene into the host bacterium.

3. A means of selecting for host cells that have

been transformed.

Genetic Engineering

Science

TL 5-1

Classroom transformation employs E. coli as the recipient of genes coding for identifiable phenotypes. The genes are carried in a plasmid vector. Two common genes used in transformation are:

1. ampr gene which codes for resistance to the antibiotic

ampicillin.

2. lacz gene which codes for beta-galactosidase, which

breaks down the lactose analog X-gal to produce a

visible blue product.

The classroom protocol uses a rapid method to render E. coli "competent" to uptake plasmid DNA containing the genes to be transformed. Although the exact mechanism of plasmid DNA by competent E. coli cells is unknown, it is believed that DNA molecules may pass through pores in the bacterial wall. Bacterial cells are treated at 0oC to crystallize the fluid membrane, preventing the electrostatic repulsion between the DNA and phospholipid membrane (both negatively charged). Freezing stabilizes the distribution of charged phosphates and addition of cations (Ca 2+) in a transformation buffer forms complexes with the exposed phosphate groups, shielding the negative charges. A plasmid molecule can then move through the pore. Heat shocking furthers the process by creating a thermal imbalance to facilitate movement of the DNA molecules.

Once plasmids have been introduced into the host cells, they must become established inside the E. coli cell. During this time the plasmid replicates and expresses the genes which will allow for selection of a transformed phenotype. Selection can be established by spreading bacteria onto a LB agar culture plate with ampicillin. Only transformed cells can survive in the medium, untransformed cells will fail to grow.

Genetic Engineering

Science

TL 5-2