Mom, Dad, and Clogged Arteries

1. Cholesterol does good things for the body, such as forming membranes and vitamin D, but it can also combine with lipoproteins to form atherosclerotic plaques in the walls of arteries.
   
   
   1. Some persons have genes that cause familial cholesterolemia.
   
   
   
   2. Gene therapy promises a way to genetically alter the cells of the liver to keep the levels of cholesterol in the more normal range.


2. For more than 3 billion years, mutation, crossing over, random gene mixing at fertilization, and hybridizations between species have contributed to the diversity of life on Earth.



3. Today, we can "engineer" genetic changes through recombinant DNA technology.
   
   
   1. DNA from different species can be cut, spliced together, and inserted into bacteria, which then multiply the DNA necessary for protein production.
   
   
   
   2. Genetic engineering has great promise for agriculture, medicine, and industry, but it has also raised ecological, social, and ethical questions.

16.1   A Toolkit For Making Recombinant DNA

1. Restriction Enzymes
   
   
   1. Bacteria possess restriction enzymes whose usual function is to cut apart foreign DNA molecules.
   
   
   
   2. Each enzyme cuts only at sites that possess a specific base sequence.
   
   
   
   3. The wide variety of restriction enzymes and their specificity makes it possible to study the genome of a particular species.


2. Modification Enzymes
   
   
   1. Many times the "sticky ends" that result from the cut can be used to pair up with another DNA fragment cut by the same enzyme.
   
   
   
   2. DNA fragments produced by restriction enzymes are treated with DNA ligase to splice the DNA fragments together to form a recombinant DNA molecule.


3. Cloning Vectors for Amplifying DNA
   
   
   1. Plasmids are circular DNA molecules in bacteria that carry only a few genes and can replicate independently of the single "main" chromosome.
   
   
   
   2. When the plasmid is replicated, any foreign DNA that might have become incorporated into it is also replicated, producing a DNA clone.
   
   
   
   3. Modified plasmids that are capable of accepting, replicating, and delivering DNA to another host cell are called cloning vectors.


4. Reverse Transcriptase to Make cDNA
   
   
   1. Even after a desired gene has been isolated and amplified, it may not be translated into functional protein by the bacteria because introns (noncoding regions) are still present.
   
   
   
   2. Researchers minimize this problem by using cDNA, which is made from "mature" mRNA transcripts.
      
      
      1. The cDNA is made from mRNA by reverse transcriptase.
      
      
      
      2. The cDNA can be inserted into a plasmid for amplification.

16.2   PCR--A Faster Way to Amplify DNA

1. The polymerase chain reaction (PCR) can be used to make millions of copies of cDNA.



2. What Are Primers?
   
   
   1. Primers are short nucleotide sequences that are made in the laboratory.
   
   
   
   2. They are recognized by DNA polymerases as the START tags for building complementary sequences of DNA dictated by computer programs stored in the machines.


3. What Are the Reaction Steps?
   
   
   1. Researchers mix primers, DNA polymerase, cellular DNA from an organism, and free nucleotides.
   
   
   
   2. Precise temperature cycles cause the DNA strands to separate, exposing the bases.
   
   
   
   3. Primers become positioned on the exposed nucleotides to form new copies of the original DNA.
   
   
   
   4. Each round of reactions doubles the number of DNA molecules to eventually produce billions of molecules from very tiny amounts of original DNA.

16.3   Focus on Bioethics: DNA Fingerprints

16.4   How Is DNA Sequenced?

1. Current laboratories use automated DNA sequencing to determine the unknown sequence of bases in a DNA sample.
   
   
   1. The machine builds DNA molecules but uses eight kinds of bases: four normal and four that are modified to fluoresce in laser light.
   
   
   
   2. When a modified base is incorporated, DNA synthesis is halted producing tagged fragments of different lengths.


2. The automated DNA sequencer separates the sets of fragments by gel electrophoresis.
   
   
   1. The "tag" base at the end of each fragment in the set is identified by the laser beam.
   
   
   
   2. The computer program in the machine assembles the information from all the nucleotides in the sample to reveal the entire DNA sequence.

16.5   From Haystacks to Needles--Isolating Genes of Interest

1. How can you isolate a particular gene for study?
   
   
   1. Create a gene library, which is a collection of bacteria that house different cloned DNA fragments, one of which is of interest.
   
   
   
   2. The library may of the entire genome or of cDNA, which is free of introns.


2. What Are Probes?
   
   
   1. DNA probes, short DNA sequences assembled from radioactive nucleotides, can pair with parts of the gene to be studied.
   
   
   
   2. This nucleic acid hybridization technique can be used with other procedures to select cells and their DNA, which may be of interest to the researcher.


3. Screening For Genes
   
   
   1. First, grow the bacterial colonies on suitable medium in a petri plate.
   
   
   
   2. Place a nylon filter over the colonies and lift some cells off.
   
   
   
   3. Place the filter in a solution to disrupt the cells but leave DNA sticking to the filter.
   
   
   
   4. Add a radioactively-labeled probe DNA to the filter where it will bind to the DNA fragments of complementary sequence.
   
   
   
   5. Expose the filter to x-ray film to locate the gene of interest, which will be in the same location as the cells in the petri plate.

16.6   Using the Genetic Scripts

1. Microorganisms can produce useful substances such as human insulin and blood-clotting factors.



2. Genetically engineered bacteria can clean up messes such as oil spills.



3. Knowing about genes may help us devise counterattacks against rapidly mutating pathogens.

16.7   Designer Plants

1. Regenerating Plants From Cultured Cells
   
   
   1. Botanists are searching the world for seeds from the wild ancestors of potatoes, corn, etc.
   
   
   
   2. The worry is that there is too little diversity in the few strains now used for food crops.
   
   
   
   3. Many plant species can be regenerated from cultured cells.
   
   
   
   4. Useful mutations, such as resistance to a toxin, can be identified.


2. How Are Genes Transferred Into Plants?
   
   
   1. An early experiment showed that a plasmid from a bacterium that normally causes tumors in plants could be modified by replacing the tumor gene with desirable genes.
   
   
   
   2. Such modified bacteria have been injected into plant cells where they expressed their "foreign" genes.
      
      
      1. Genetically modified crop plants could increase food production or grow with greater resistance to pest attack.
      
      
      
      2. Genetically engineered plants may also produce human hemoglobin, melanin even plastics!

16.8   Gene Transfers in Animals

1. Supermice and Biotech Barnyards
   
   
   1. In 1982, the rat gene for somatotropin production was introduced into mouse eggs; the mice which subsequently expressed the rat gene grew larger than their littermates.
   
   
   
   2. Farm animals may be used to produce TPA for diminishing the severity of heart attacks or CFTR used in the treatment of cystic fibrosis.
   
   
   
   3. Cloning of animals could lead to disease-resistant types.


2. Mapping and Using the Human Genome
   
   
   1. The Human Genome Initiative is dependent on this technology.
   
   
   
   2. The information gained will give insights into genetic disorders and ultimately, provide for gene therapy.
   
   
   
   3. The new field of genomics will be concerned with mapping and sequencing the genomes as well as elucidating the possible evolutionary relationships of groups of organisms.

16.9   Safety Issues

1. Genetically engineered bacteria have "fail-safe" genes included in the DNA which are supposed to be lethal if the bacteria escapes into a non-lab environment.



2. The general public is concerned about organisms being released that are not "natural" and may endanger human lives.

16.10   Biotechnology in a Brave New World

1. Microarrays, or gene chips, can reveal a stunning amount of information about an individual's DNA.



2. Who Gets Well?
   
   
   1. Gene therapy has been successful in a trial against SCID-X1.
   
   
   
   2. Which disorders will receive attention--and money?


3. Who Gets Enhanced?
   
   
   1. Eugenic engineering is idea of being able to select desirable human traits.
   
   
   
   2. Who decides what is "desirable?"


4. Send In the Clones? Don't Bother, They Are Here
   
   
   1. Xenotransplantation is the transferring of an organ from one species to another.
   
   
   
   2. Pigs can be engineered to lack certain genes that would cause rejection problems when their organs are transplanted to humans.


5. Weighing the Benefits and Risks
   
   
   1. Some say we should never alter the DNA of any organism; others say we already have.
   
   
   
   2. The question for the future seems to be not whether we will perform these changes but by how much.

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