Synthetic genes

SIGNIFICANCE: Synthetic genes have been shown to function in biological organisms. Scientists hope that it will prove possible to restore normal function in diseased humans, animals, and plants by replacing defective natural genes with appropriately modified synthetic genes.

A Brief History

In 1871, Swiss physician Johann Friedrich Miescher reported that the chief constituent of the cell nucleus was nucleoprotein, or nuclein. Later it was established that the nuclei of bacteria contained little or no protein, so the hereditary material was named nucleic acid. At the end of the nineteenth century, German biochemist Albrecht Kossel identified the four nitrogenous bases: the purines and and the pyrimidines and uracil (U). In the 1920s, Phoebus A. Levene and others indicated the existence of two kinds of nucleic acid: and deoxyribonucleic acid (DNA); the latter contains instead of uracil.

The chemical identity of genes began to unfold in 1928, when Frederick Griffith discovered the phenomenon of genetic transformation. Oswald Avery, Colin MacLeod, and Maclyn McCarty (in 1944) and Alfred Hershey and Martha Chase (in 1952) demonstrated that DNA was the hereditary material. Following the elucidation of the structure of DNA in 1953 by James Watson and Francis Crick, pioneering efforts by several scientists led to the eventual synthesis of a gene. The successful enzymatic synthesis of DNA in vitro (in the test tube) in 1956, by Arthur Kornberg and colleagues, and that of RNA by Marianne Grunberg-Manago and Severo Ochoa, also contributed to the development of synthetic genes. In 1961, Marshall Nirenberg and Heinrich Matthaei synthesized polyphenylalanine chains using a synthetic messenger RNA (mRNA). In 1965, Robert W. Holley and colleagues determined the complete sequence of alanine isolated from yeast. The interpretation of the by several groups of scientists throughout the 1960s was also clearly important.

In 1970, Har Gobind Khorana, along with twelve associates, synthesized the first gene: the gene for an alanine in yeast. There were no automatic DNA synthesizers available then. In 1976, Khorana’s group synthesized the tyrosine suppressor tRNA gene of Escherichia coli (E. coli). The lac operator gene (twenty-one nucleotides long) was also synthesized, introduced into E. coli, and demonstrated to be functional. It took ten years to synthesize the first gene; by the mid-1990s, gene machines could synthesize a gene in hours.

Gene Synthesis

Protein engineering is possible by making targeted changes in a DNA sequence to produce a different product (protein) with different properties, such as stress tolerance. The process of targeting a specific change in the sequence (site-directed mutagenesis) allows the correlation of gene structure with protein function. Rapid sequencing with modern capillary DNA sequencers facilitates determination of the order of nucleotides that make up a gene in a matter of hours.

Once the sequence of a gene is known, it can be synthesized from nucleotides using gene machines. A gene machine is simply a chemical synthesizer made up of tubes, valves, and pumps that bonds nucleotides together in the right order under the direction of a computer. An intelligent person with a minimum of training can produce synthetic genes. A gene may be isolated from an organism using restriction enzymes (any of the several enzymes found in bacteria that serve to chop up the DNA of invading viruses), or it may be made on a gene machine. For example, the chymosin gene (an used in cheese making) in calves can be synthesized from its known nucleotide sequence instead of isolating it from calf DNA using restriction enzymes. Alternatively, chymosin can be obtained from calf stomach cells, which can be transformed into DNA through reverse transcription.

New or modified genes may be manufactured to obtain a desired product. Gene synthesis, coupled with automated rapid sequencing and protein analysis, has yielded remarkable dividends in medicine and agriculture. For example, researchers have influenced plants' biological processes to direct them to grow more effectively. Genetic engineers are designing new proteins from scratch to learn more about protein function and architecture. With synthetic genes, the process of mutagenesis can be explored in greater depth. It is possible to produce various alterations at will in the nucleotide sequence of a gene and observe their effects on protein function. Such studies carry the potential to unravel many biochemical and genetic pathways that could be the key to a better understanding of health and disease.

Key Terms

• restriction enzymean enzyme that cleaves, or cuts, DNA at specific sites with sequences recognized by the enzyme; also called restriction endonucleases
• reverse transcriptionthe synthesis of DNA from RNA

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