Recombinant

Since its inception in the mid-1970s, recombinant DNA (rDNA)67-74 (genetic engineering) technology has driven much of the fundamental research and practical development of novel drug molecules and proteins. rDNA technology provides the ability to isolate genetic material from any source and insert it into cells (plant, fungal, bacterial, animal) and even live animals and plants, where it is expressed as part of the receiving organism's genome. Before discussing techniques of genetic engineering, a review of some of the basics of cellular nucleic acid and protein chemistry is relevant.75-77

Most of the components that contribute to cellular homeostasis are proteins—so much so that more than half of the dry weight of a cell is protein. Histones, cellular enzymes, membrane transport systems, and immunoglobulins are just a few examples of the proteins that carry out the biological functions of a living human cell. Proteins are hydrated three-dimensional structures, but at their most basic level, they are composed of linear sequences of amino acids that fold to create the spatial characteristics of the protein. These linear sequences are called the primary structure of the protein, and they are encoded from DNA through RNA. The information flow sequence DNA ^ RNA ^ protein has, for many years, been called the biological "Central Dogma."78,79 The specific sequence of amino acids is encoded in genes. Genes are discrete segments of linear DNA that compose the chromosomes in the nucleus of a cell. The Human Genome Project has revealed that there are between 30,000 and 35,000 functional genes in a human, encompassing about 3,400,000,000 base pairs (bp).80

As depicted in Figure 4.4, in the nucleus of the cell, double-stranded DNA undergoes a process of transcription (catalyzed by RNA polymerase) to yield a single-stranded molecule of pre-mRNA. Endonucleases then excise nonfunctional RNA sequences called introns, from the pre-mRNA, to yield functional mRNA. In the cytoplasm, mRNA complexes with the ribosomes and the codons are read and translated into proteins. The process of protein synthesis in Escherichia coli begins with the activation of amino acids as aminoacyl-transfer RNA (tRNA) derivatives. All 20 of the amino acids undergo this activation, an adenosine triphosphate (ATP)-dependent step catalyzed by aminoacyl-tRNA synthetase. Initiation involves the mRNA template, N-formylmethionyl tRNA, the initiation codon (AUG), initiation factors, and the ribosomal subunits. Elongation occurs (using several elongation factors) with the aminoacyl-tRNAs being selected by recognition of their specific codons and forming new peptide bonds with neighboring amino acids. When biosynthesis of the specific protein is finished, a termination codon in the mRNA is recognized, and release factors disengage the protein from the elongation complex. Finally, the protein is folded and post-translational processing occurs.81,82 Processes that might be used in this step include removal of initiating residues and signaling sequences, proteolysis, modification of terminal residues, and attachment of phosphate, methyl, carboxyl, sulfate, carbohydrate, or prosthetic groups that help the protein achieve its final three-dimensional shape. Specialized

chaperone proteins can also direct the three-dimensional formation. Posttranslational modifications occur in mammalian cells in the endoplasmic reticulum or the Golgi apparatus before the protein is transported out of the cell. Most posttranslational modifications occur only in higher organisms, not in bacteria. The three-base genetic codon system is well known and has been conserved among all organisms. This allows rDNA procedures to work and facilitates the development of a model for the amino acid sequence of a protein by correlation with the codon sequence of the genome.

Recombinant DNA Technology

The fundamental techniques involved in working with rDNA include isolating or copying a gene; inserting the precise gene into a transmissible vector that can be transcribed, amplified, and propagated by a host cell's biochemical machinery; transferring it to that host cell; and facilitating the transcription into mRNA and translation into proteins. Cloned DNA can also be removed or altered by using an appropriate restriction endonuclease. Because genes encode the language of proteins, in theory, it is possible to create any protein if one can obtain a copy of the corresponding gene. rDNA methods require the following83:

• An efficient method for cleaving and rejoining phosphodi-ester bonds on fragments of DNA (genes) derived from an array of different sources

• Suitable vectors or carriers capable of replicating both themselves and the foreign DNA linked to them

• A means of introducing the rDNA into a bacterial, yeast, plant, or mammalian cell

• Procedures for screening and selecting a clone of cells that has acquired the rDNA molecule from a large population of cells

There are two primary methods for cloning DNA84 using genomic and cDNA libraries as the primary sources of DNA fragments, which, respectively, represent either the chromosomal DNA of a particular organism or the cDNA prepared from mRNA present in a given cell, tissue, or organ. In the first method, a library of DNA fragments is created from a cell's genome, which represents all of the genes present. The library is then screened against special DNA probes. Lysing the genomic contents to generate fragments of different sizes and compositions, some of which should contain the genetic sequences that encode the specific activity that one is seeking, creates the library. With knowledge of the protein sequence that the gene specifies, DNA probes can be synthesized that should hybridize with corresponding fragments in the library. By labeling the probes with fluorescent or radioactive tags, probe molecules that hybridize and form double-helical DNA can be identified and isolated elec-trophoretically. The DNA from the library can then be amplified by a technique such as the polymerase chain reaction (PCR), inserted into a vector, and transferred into a host cell. A comparison of these methods is given in Table 4.1.

The second major method for cloning DNA represents only genes that are being expressed84 at a given time and involves first the isolation of the mRNA that encodes the amino acid sequence of the protein of interest. Treating the mRNA with the viral enzyme reverse transcriptase in the presence of nucleoside triphosphates (NTPs) causes a strand of DNA to be synthesized complementary to the mRNA matrix, affording

TABLE 4.1 Characteristics of Genomic versus cDNA Libraries

Characteristic

Genomic

cDNA

Source of genetic

Genomic DNA

Cell or tissue

material

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Blood Pressure Health

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