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Biology

Chapter 12: Gene Technology

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Lecture

Cloning and Modifying the Genetics of Organisms

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Viruses, Bacteria, Plasmids and Vectors

Gene technology grew out of studies of viruses attacking bacteria. We tend to think of both organisms as dangerous and harmful, but most bacteria are either benign (harmless) or beneficial, like the E. coli strains that help you digest your food. Some forms of viral infections can damage these helpful bacteria, so study of the effects of such phages (viral bacteria-killers) on bacteria was important and necessary for the treatment of certain diseases.

Viruses and Vectors

Gene technology grew out of studies of viruses attacking bacteria. We tend to think of both organisms as dangerous and harmful, but most bacteria are either benign (harmless) or beneficial, like the E. coli strains that help you digest your food. Some forms of viral infections can damage these helpful bacteria, so study of the effects of such phages (viral bacteria-killers) on bacteria was important and necessary for the treatment of certain diseases.

To see how DNA tranformation takes place, use the DNA Transformation 1 animation at the Gene Almanac. Click on "Play" (you will need Macromedia Flash on your browser for this to work. You can download the animation and play it locally as well -- this is a good idea if you want to review it later.

  • Who first successfully recombined genes from different bacteria?
  • What genes did they introduce into bacteria? Why?
  • What is heat shock? What happens when bacteria are subjected to heat shock?
  • How is transformation of a gene different from meiosis cross-over?

An unexpected side effect of this research, and one which is changing our lives in many ways, was the discovery of the mechanisms of gene cutting and cloning. Both bacteria or prokaryotic cells and the eukaryotic cells which make up multicellular organisms have special enzymes designed to identify and cut up DNA — we've seen them already at work in mRNA transcription. They also cut up foreign DNA into harmless bits. These enzymes are called restriction enzymes because they restrict the ability of foreign DNA to replicate and affect the host cell. Bacteriologists and geneticists discovered how to isolate these enzymes and use them to cut DNA at targeted points.

A restriction enzyme looks for a specific sequence, which can vary (depending on the enzyme) from 4 nucleotides (one codon + one nucleotide) to 23 nucleotides long. Obviously, those restriction enzymes which match longer sequences are less likely to find a restriction site in any randomly chosen DNA fragment than the enzymes which match shorter sequences. One requirement for any recognition sequence is that it be a palindrome: the sequence must read the same way in both directions on the double helix.

DNA palindrome For example, the DNA strand GATATC will work, since the upper and lower strands read the same when both are read from the 5' to the 3' direction.
When the restriction enzyme cuts the strand, it breaks only the covalent bonds between the backbones of phosphate ions and ribose between a nucleotide pair -- in this case, we'll assume between the G and A nucleotides. DNA Restriction
DNA sticky ends The DNA helix is still held together by hydrogen bonds, but these are so weak that the strands separate, each with a G on its own, and then with a sequence of CTATA on the opposite side.

The exposed nucleotides still have the capacity to form hydrogen bonds, which makes them "sticky" around other nucleotides or sequences of DNA which may match the exposed ends.

When a new DNA sequence with the same sticky ends meets up with the DNA fragment, the process is reversed. The hydrogen bonds form, then the covalent bonds are recreated by another enzyme called DNA ligase. This enzyme is always present in living cells, since it is the enzyme which constructs the single continuous strand of DNA from component strands to match the 5' to 3' strand during DNA replication. It constantly goes around the nucleus (of eukaryotes) or the DNA ring of prokaryotes, repairing broken DNA--even foreign DNA which has managed to find a sticky end in the host DNA material.

You can review just the DNA restriction process at the Gene Almanac. This animation allows you to step through as an EcoRI restriction enzyme cuts E. coli DNA at the GAATTC marker.

Gene libraries

Once geneticists understood how restriction enzymes work, they began looking for them and cataloguing which enzymes cut which sequences. By using the appropriate restriction enzymes, a geneticist can cut up all the DNA in a cell and create a library of gene fragments. By studying the fragments, the geneticists can map the entire DNA chromosome content, or genome, of an organism. Such a library of original DNA fragments, including introns and genes which may no longer be expressed, is a genome library.

Once the genome library has been created, geneticists can select specific DNA segments, then create complimentary mRNA containing only active exon sequences. Then they can use a process very similar to that used by viral agents to create virus-based DNA inside a host cell. In an infected host cell, mRNA of course exists in cytoplasm containing nutrients, enzymes, and the raw materials for both DNA and RNA. In the lab, the synthesized mRNA is introduced into a nucleotide soup which also contains the enzyme reverse transcriptase. The enzyme goes to work, matching nucleotides to the mRNA until a single DNA strand has been created. The original mRNA is degraded, or broken down into base components that are washed out of the mixture. The reverse transcriptase then "reads" the single DNA strand that it has created, and with the help of DNA ligase creates the matching DNA strand. When they finish. a clean double-helix strand of DNA has been created of all the active genes in the segment, but lacking all the introns. If all the mRNA produced by a cell is used to create DNA strands, the outcome is a complementary DNA or cDNA library of the active genes. (See the short reverse transcriptase animation at Rutgers for more details on how this happens).

Both genomic and cDNA libraries are useful. A genome library allows us to study the entire structure of the chromosome. A complementary DNA library allows us to study particular genes in detail, and also gives us a template to use with bacteria. Since bacteria don't perform all the functions that eukaryotic cells perform, they lack a number of enzymes, including those that splice mRNA segments together when an intron has interrupted a gene sequence. By introducing only cDNA (made from mRNA) into the bacteria, we increase the chance that the bacteria can replicate a viable eukaryotic gene.

The human genome project is an example of a cDNA library creation. Information from mapping the human genome is available at many public sites, since the work was done largely with public moneys under the auspices of the Department of Energy of the United States. You can look up a specific gene, for example, the gene whose defect causes Huntington Disease, which we discussed some time ago in our study of inheritance. The Genetic Home Reference page for Huntington's Gene (hg) describes the normal function of the gene and the mutation that causes inheritable Huntington Disease. How does the mutation alter normal cell function?