ABSTRACTS:

ARTICLE TITLE: Targeted Gene Replacement
AUTHOR: Mario R. Capecchi
JOURNAL: SCIENTIFIC AMERICAN, March 1994, pages 52-59
ABSTRACT: A technique for changing nucleotide sequences within living cells of mammals promises to be a powerful tool for understanding the workings of specific genes. By rewriting the genetic instructions (nucleotide sequences) within an organism we can gain insight into the effect such changes have upon the developing or adult animal. Proteins carry out most of the biochemical operations within the cells. Knowing the sequence of amino acids that make up a particular protein is useful information but that sequence alone can not predict the utility and function of that protein throughout the life of the animal. The technique of targeted replacement of genes and segments of DNA allows us to study the precise effects of those genes within the animal.

Gene targeting permits investigators to determine how and when genes work within a developing as well as an adult animal. Traditional genetic studies involve the use of prolific bacteria, such as Escherichia coli, or tiny animals, such as Caenorhabditis elegans (a soil worm) or Drosophila melanogaster (a fruit fly). These small organisms multiply rapidly and are easily housed within a laboratory. Working with comparatively larger animals, such as mice, requires techniques that provide genetic information within a few generations using only a few individuals. The diploid nature of most mammalian cells (having pairs of chromosome and, hence, pairs of genes) protects the individual from succumbing to the effects of a single mutation. The presence of a second gene may help balance and ameliorate the effects of the mutated gene. One of the problems that we face using whole animals as our models for genetic investigations is that most of the mutations that allow the animal to survive to the adult stage are, by definition, benign and not life threatening. Using the traditional models we really have no access to the mechanisms of genetic alterations that may have profound effects upon the adult form of the model. The technique of gene targeting involves creating a synthetic gene and flanking that gene with specific sections of normal DNA. Once these sections are introduced into a living cell, they align themselves within the cell along the similar section of the cell's chromosome. The modified gene is inserted into the existing chromosome. Likewise, genes may be removed by reversing this process. The effects of gene removal or gene modification may then be studied as these cells grow and develop within an intact animal. Embryo-derived stem cells (taken from the early stages of development) can be cultured in the laboratory and have the ability to become any type of tissue cells. Once mutated, they may be incorporated into a mouse embryo to carry their modified genetic blueprint to the developing mouse. This technique enables scientists to study the more than 5,000 disorders attributed to genetic defects and mutations.

KEY WORDS/PHRASES
Embryo-derived stem cells
Gene targeting
Genetic alterations
Synthetic gene

ARTICLE TITLE: Breaking the Shackles of the Genetic Code: Engineering Retroviral Proteases Through Total Chemical Synthesis
AUTHORS: Stephan B.H. Kent, Manuel Baca, John Elder, Maria Miller, Raymond Milton, Saskia Milton, J.K.M. Rao, and Martina Schnolzer
JOURNAL: ADVANCES IN EXPERIMENTAL BIOLOGY, Volume 362, 1995, pages 425-438
ABSTRACT: This review describes four biochemical advances that permit the close study of enzyme actions. Protein enzymes are essentially the machines of any living cell. Enzymes conduct and catalyze almost every chemical reaction within the cell. Recent advances in the techniques of recombinant DNA molecular biology have added to the tools available to aid the study of enzyme chemistry. Enzymes, as well as other proteins may now be manufactured almost at will. Techniques, such as those described in this report, can be used to make proteins with specific amino acid sequences and a host of variant side-chain compounds. These proteins, DNA, RNA, and enzymes are then used to study normal cellular biochemical function.

The powerful new tools described include: (a) the exact measurement of protein weight, (b) the ability to study complex protein conglomerates with mass spectroscopy, (c) a rapid protein ladder sequencing method for protein synthesis, and (d) a new synthesis method that allows for the complete synthesis of very complex proteins. With these processes every aspect of the manufacture of a protein molecule can be controlled, including the positioning and sequencing of amino acids and their functional side-chain groups. These techniques allow molecular biologists to conduct experiments with the same precision that has been enjoyed by traditional chemists for generations. By way of example the development of a completely synthetic protease (an enzyme) component of the Feline Immunodeficiency Virus (FIV) is presented to explain the steps in this process. The examples and discussion within this review show that protein chemistry, specifically enzyme chemistry and by extension all DNA, and RNA chemistry is now amenable to standard laboratory manipulation. Precise backbone modifications may be made. Specific chemical geometries may be introduced to dictate the folding of these huge bio-molecules.

KEY WORDS/PHRASES
Enzyme actions
Feline Immunodeficiency Virus
Protein chemistry
Protein synthesis
Synthetic protease