Fundamentals of Biochemistry - A. A. Anisimov 1986

Nucleic Acids
Genetic Engineering

A long-standing dream of geneticists and biochemists has been to create living organisms with pre-determined hereditary traits and a specific METABOLISM. This is the exact challenge undertaken by a new scientific field: Introduction/32.html">Genetic Engineering. Its primary objective is the in vitro production of recombinant DNA molecules, their Replication, and introduction into organisms to impart new hereditary characteristics. Genetic engineering is built upon the universality of genetic material, which makes it possible to construct recombinant DNA molecules from the DNA of different organisms—such as bacterial and Eukaryotic Cells—and introduce these constructs into living cells. Although genetic engineering is still in its infancy, its achievements are already striking. It paves the way for the rapid creation of highly productive industrial bacterial strains, the improvement of crop yields, and the Treatment of human Genetic Disorders. Building upon this foundation, several practically significant projects have been developed, such as introducing genes responsible for atmospheric Nitrogen Fixation into agricultural plants. Definite progress has been made in this direction: the nitrogen-fixing Operon genes from Klebsiella Bacteria have been successfully transferred into E. coli cells. Another major undertaking is the introduction of normal genes into the cells of individuals suffering from Hereditary diseases.

Currently, Genetic engineering Methods are successfully applied to create bacterial strains that produce biologically active compounds, including Hormones (Insulin, Somatostatin) and the antiviral agent interferon. Furthermore, the advancement of genetic engineering has made it possible to study the Structural and functional features of eukaryotic genetic material.

Conducting genetic engineering research first requires obtaining specific DNA fragments, i.e., genes. Several methods are employed for this purpose. The earliest genes were synthesized chemically. In 1969, H. Khorana's research group synthesized the Yeast Alanine tRNA Gene, for which the 77-nucleotide sequence had been fully deciphered by that time. Small DNA fragments (ranging from 4 to 13 Base Pairs) were chemically synthesized and subsequently joined in the required order using ligase. The resulting gene lacked regulatory regions and was functionally inactive.

In 1976, the same laboratory synthesized an E. coli DNA segment encoding Tyrosine suppressor tRNA. This tRNA gene consists of 126 base pairs, flanked on one end by a 52-base-pair promoter region and on the other by a 21-base-pair terminator region, with AATT and TTAA tetranucleotides attached to the ends of the segment. The synthetic gene proved to be fully active. After introducing this gene into a mutant T4 bacteriophage strain that lacked it, the bacteriophage reproduced successfully in E. coli cells, thereby becoming fully viable.

G. Boyer's group chemically synthesized the gene for the hormone somatostatin. This synthesis was carried out based on the Amino Acid Sequence of the polypeptide. The triplets corresponding to these Amino Acids were chemically synthesized and linked together. The resulting double-stranded DNA gene was inserted into the E. coli genome adjacent to the ß-galactosidase gene. As a result, the bacterium began to produce a fusion protein consisting of ß-galactosidase on one end and somatostatin on the other. These brilliant results demonstrated the feasibility of chemically creating genes indistinguishable from natural ones.

Later on, gene synthesis shifted toward a less labor-intensive and faster method: synthesis using Reverse Transcriptase. Studies of this enzyme demonstrated that any RNA, even a synthetic polyribonucleotide, can serve as a template for DNA formation. This breakthrough opened the way for synthesizing A wide variety of genes using messenger RNAs (mRNAs). The process begins with the purification of the mRNA corresponding to a specific gene. Using this mRNA as a template, reverse transcriptase synthesizes a complementary DNA (cDNA) copy.

Using this approach, laboratories worldwide—including in the USSR—have synthesized genes encoding human, rabbit, and mouse Globins, bovine lens protein, as well as genes from the vaccinia virus and certain Bacteriophages. However, it should be noted that using mRNA as a DNA Synthesis template yields only the structural, coding portion of the gene, while the regulatory regions required for the proper functioning of many genes remain absent. Moreover, eukaryotic genes have a complex Structure, sometimes consisting of multiple separate segments located in different PARTS OF THE genome. This imposes limitations on The Use of the aforementioned method. Consequently, the isolation of natural genes directly from The Genome is widely practiced. For this purpose, DNA is cleaved, and the DNA fragment of interest is incorporated into a vector, which allows the target fragment to be replicated in numerous copies and introduced into recipient cells.

A vector is a DNA molecule capable of transferring foreign DNA of any origin into a Cell and ensuring its subsequent replication there. Vectors capable of autonomous replication are widely used. These typically include temperate bacteriophages or, more frequently, Plasmids. Such vectors make it possible to obtain numerous copies of a foreign gene. They are utilized for cloning—that is, obtaining a homogeneous population of DNA molecules. Homogeneity is ensured because all molecules are direct descendants of a single DNA molecule.

The incorporation of a foreign DNA fragment into a plasmid is performed in vitro. First, both components are converted into a linear form. Higher Organism DNA is usually fragmented using restriction Enzymes (Restriction Endonucleases). Certain restriction enzymes generate DNA fragments with "sticky" ends. The base complementarity of these sticky ends allows any DNA fragments generated by the same restriction enzyme to be joined together, making it possible to insert a DNA fragment into a vector. If a DNA fragment has "blunt" ends, polynucleotide transferase can be used to attach runs of adenylyl and thymidylyl NUCLEOTIDES to them. The length of these poly(A) and poly(T) "sticky" tails is 50–100 nucleotides, which is sufficient for The formation of hybrid structures between two different DNA molecules.

The vector is typically "opened" using the same restriction enzyme that was used to generate the DNA fragment. Conditions are specifically optimized to cleave the vector at only one precise site. The joining of the DNA fragment and the vector is usually achieved via their sticky ends, and the phosphodiester bonds between adjacent nucleotides are sealed using DNA ligase.

In some cases, DNA molecules are joined at their blunt ends using a specialized enzyme. Frequently, linkers and adaptors—short DNA fragments engineered with an end corresponding to one restriction enzyme on one side and a different restriction enzyme on the other—are employed to connect DNA molecules. This facilitates the joining of DNA fragments, resulting in the formation of a recombinant DNA molecule. Such a molecule contains the complete set of plasmid genes required for autonomous replication alongside the foreign DNA.

Depending on the objectives and goals of the experiments, various plasmids are used in genetic engineering, though most are constructed in vitro from plasmid fragments of diverse origins. Plasmids selected for this purpose are cleaved by a specific restriction enzyme at only a single site and replicate rapidly, yielding 1,000–3,000 copies per cell. Cloning with these plasmids produces specific, highly purified DNA fragments in large (milligram) quantities (Fig. 4.22). The resulting recombinant DNA molecule is introduced into specially treated bacteria. To select for bacteria that have successfully taken up the vector, the latter is marked by incorporating an Antibiotic Resistance gene.

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Fig. 4.22. Scheme of a genetic engineering experiment (after S. G. Inge-Vechtomov, 1983):

1 — plasmid treated with restriction enzyme, 2 — DNA fragments treated with the same restriction enzyme, 3 — chimeric plasmid, 4 — transformed bacterium, 5 — transformed daughter cells

Because isolating and purifying individual genes is extremely complex and labor-intensive, the "shotgun" cloning method—which bypasses this initial tedious step—has gained widespread use. The Procedure begins by fragmenting DNA containing the desired gene either mechanically or via restriction enzymes. These fragments are then joined with vector DNA molecules (such as plasmids) that have been linearized and provided with sticky ends using restriction enzymes. The resulting hybrid molecules are introduced into E. coli cells, after which the bacteria carrying the target gene are identified. By screening a vast number of bacterial clones harboring random DNA fragments, the sought-after gene is guaranteed to be present in one of the clones. First, bacterial clones that successfully took up plasmids are selected. If the plasmid carries a gene for resistance to a specific antibiotic, the bacteria are plated on a medium containing that antibiotic, allowing only those that received the plasmid to survive. Subsequently, the gene of interest must be identified.

Genes encoding enzymes involved in low-molecular-weight metabolism are easily detected. If an introduced plasmid-borne gene Functions within the host cell, bacterial clones containing the plasmid with this gene will grow on selective media. However, natural genes from higher and lower eukaryotes rarely express themselves in E. coli. To detect them, researchers often utilize vectors resistant to two Antibiotics, such as streptomycin and ampicillin, and insert the cloned gene directly into one of the resistance genes. Bacterial clones containing the recombinant plasmid are then identified by the loss of that specific resistance.

Alternatively, a clone carrying the DNA of the gene under study can be identified through the nucleic acid Hybridization capacity. To this end, mRNA transcribed from that gene is frequently utilized. Another approach is hybridization with a DNA probe—a single-stranded DNA fragment whose nucleotide sequence closely matches one of the strands of the cloned gene. Because the DNA probe is complementary to the second strand, it specifically hybridizes with the target DNA. If the DNA probe is labeled with a radioactive isotope, the bacterial clone containing the desired DNA fragment can be detected using autoradiography.

The final stage in genetic engineering projects involves adapting the introduced gene to its new genetic and physiological environment and achieving its expression—that is, the Synthesis of the specific protein. Introducing eukaryotic DNA into a bacterial cell genome presents several challenges. To ensure the functioning of the inserted gene, a "strong," highly efficient promoter is placed immediately upstream of it in the plasmid. Another technique, pioneered by H. Khorana, is Gene Fusion. Start and stop signals are excised from the boundaries between genes, allowing any genes to be fused and their protein products combined. The resulting Polypeptides generally fold into their tertiary structure independently, and the peptide bridge linking the products of different genes can subsequently be cleaved by specific means.

For instance, the gene encoding the peptide hormone somatostatin was artificially synthesized and lacked regulatory regions. To ensure its expression in E. coli, it was inserted into a vector alongside a well-characterized E. coli gene encoding ß-galactosidase. The nucleotide sequence of the synthetic gene was transcribed and then translated as the C-terminal fragment of ß-galactosidase. The synthesized active somatostatin was separated from ß-galactosidase by cleaving the polypeptide chain with a specific reagent. Thus, the regulatory signals of the ß-galactosidase gene were co-opted to drive the expression of the synthetic gene.

In recent years, attempts have been made to employ novel vectors in genetic engineering. For example, recombinant vectors capable of integrating a foreign DNA fragment directly into the host chromosome have found application. These include certain chromosomal DNA fragments from Bac. subtilis, as well as a fragment of the temperate phage λ that encodes the integration enzyme. The use of these vectors immediately yields transformed cells, which is extremely advantageous; however, the yield of such cells remains low.

Efforts are also underway to utilize eukaryotic vectors based on Oncogenic Viruses. Plasmids discovered in lower eukaryotic cells—such as yeast—can also serve as vectors. The application of these novel vectors opens up promising Prospects for working with eukaryotic cells.

Significant and fascinating research in genetic engineering is being conducted in the Soviet Union under the leadership of Academics A. A. Baev and Yu. A. Ovchinnikov. The Institute of Bioorganic Chemistry of the USSR Academy of Sciences has developed bacterial strains that produce various hormonal preparations, and work is nearing completion on obtaining highly productive bacteria capable of synthesizing critical medical substances such as human insulin and interferon.

The synthesis of human a-F interferon by genetically engineered E. coli strains was the result of massive efforts led by Yu. A. Ovchinnikov and his coworkers. Total interferon mRNA was isolated from human Blood Leukocytes (yielding 400 µg of mRNA from 10⁵ leukocyte cells). This mRNA served as a template for reverse Transcription, producing a single-stranded DNA molecule 650–900 nucleotides in length.

The second DNA strand was synthesized using DNA polymerase I, yielding double-stranded DNA—the human leukocyte interferon gene. Linkers were chemically attached to this gene, enabling its insertion into a restriction enzyme-cleaved plasmid. These plasmids were then introduced into E. coli cells. Colonies containing plasmids with the inserted DNA were selected via hybridization with a synthetic oligonucleotide radiolabeled with ³²P.

Out of 30,000 colonies, 40 were selected for further analysis to identify the clones containing the complete gene. Subsequently, employing a range of molecular biology and genetic engineering techniques, a single bacterial clone was chosen. From this clone, a recombinant plasmid was isolated, yielding a 575-nucleotide DNA fragment. Sequencing revealed that The structure of this fragment was almost identical to that of the human leukocyte interferon F gene.

To ensure expression of the interferon gene in E. coli cells, it had to be equipped with transcriptional and translational regulatory elements. These regulatory regions were also obtained using various genetic engineering methods (restriction enzyme Digestion, cloning, etc.) and then ligated to the interferon gene. As a result, a series of recombinant E. coli clones carrying the IFN—F gene under the control of different promoters was generated. The Tryptophan promoter proved to be the most efficient. Extracts derived from these bacteria demonstrated high antiviral activity.

Under the leadership of V. G. Debabov, the world's first microbial strain synthesizing large quantities of the essential amino acid Threonine was developed. Genetic engineering methods made it possible to achieve this in just three years, whereas conventional methods would require several decades to obtain new strains. The resulting strain produces 30 g/L of threonine in 24 hours.

In ROOT nodule bacteria, the capacity for Nitrogen fixation is determined by the plasmid genome (with only partial chromosomal control), while host-plant Specificity is likewise governed by a plasmid. In recent years, utilizing genetic engineering techniques—for which plasmids serve as a convenient vehicle—researchers have successfully significantly enhanced the nitrogen-fixing capacity of Rhizobium and induced nodule formation in agricultural plants that previously lacked this ability.

In 1972, the nitrogen fixation gene was successfully introduced into E. coli, and recent years have marked the beginning of genetic engineering efforts in crop plants aimed at introducing the nitrogen-fixation gene into them. Agrobacteria harbor oncogenic Ti plasmids (T-DNA), which integrate into the host genome, inducing crown gall tumors. Through tissue culture techniques, fully functional plants harboring T-DNA in their genome can be regenerated from such tumors. Researchers have successfully inserted various specific genes into T-DNA, allowing them to be detected in the regenerated plants. This approach is currently being explored to introduce the nitrogen fixation gene into cereal crops.

The breakthroughs achieved in genetic engineering empower scientists to manipulate genomes and cross interspecies barriers, opening up possibilities for gene transfer between organisms that have historically virtually never engaged in genetic exchange. Nevertheless, one must bear in mind that manipulating certain genes can lead to The Emergence of hazardous organisms with unpredictable infectivity and ecological traits. For instance, one can envision a scenario where an E. coli strain carrying a plasmid inserted with the genome of a human pathogenic virus colonizes the human gut, potentially precipitating the spread of the disease. In response to these concerns, an international conference was held in 1975 to discuss and prevent potential adverse consequences arising from the uncontrolled use of genetic engineering. The participants agreed that recombinant DNA research should continue, provided that biological and physical containment barriers are implemented to prevent the dissemination of newly created organisms. Specifically, experiments of a certain category are restricted to the use and creation of bacteria and plasmids incapable of surviving outside the laboratory (e.g., dying at temperatures above +35°C, such as within The Human Body).