by Academician A. BAYEV, Academic Secretary, Department of Biochemistry, Biophysics and Chemistry of Physiologically Active Compounds, USSR Academy of Sciences

The scientific and technological revolution is spreading fast-to biology as well. It has become possible to make headway in the cognition of vital processes thanks to the marriage of biology with chemistry and physics enabling biologists to gain a "chemical mentality". The hard sciences, by taking up an area cardinally different from their proper domain, have exhibited their universality and midwifed the birth of a variety of crossover disciplines, such as biochemistry and biophysics, molecular biology and genetics as well as bioorganic chemistry - all that known under the coverall name of "physicochemical biology".

The essentially new method of genetic and cell engineering now in a biologist's arsenal has paved the way for the rapid development of biotechnology and its hands-on uses. Economists say that by the year 2000 biotechnological industries worldwide are to turn out products to the tune of 40 bln dollars and more.

For its part, biotechnology stimulates further progress in genetic (gene) engineering. The very term sounds like a malaprop, with "gene", the basic unit of heredity, the holy of holies of life, rubbing shoulders with the rigorous technical word "engineering". And yet the term is very exact in conveying the substance of the method. Gene engineering is a conscious and purposeful designing ("engineering") of functionally active genetic structures, i.e. those capable of performing outside the host organism. Such structures are called "recombinant DNAs". In other words, gene engineering implies the designing and reproduction of artificial programs of heredity.

A live cell may be compared to a miniature chemical plant with topnotch technology. Nothing extra, nothing superfluous, all the processes are in trim, rigorously consistent with a thoroughly verified hereditary program of DNA molecules. There is a "control center" responsible for "production target reaching", i.e. the synthesis of all the various genes. Thus each block (gene) is responsible for a definite product charged with specified functions in the cell. Clearly, by interfering in a genetic program and introducing new information in the form of recombinant DNA molecules, a biologist can design an organism in keeping with his plan.

An ability to isolate and transplant genes is a scientific wonder indeed. This accomplishment has been made possible owing to the progress of physicochemical biology, in particular, to the achievements of the chemistry of enzymes and of the chemistry of nucleic acids. It is biochemistry that has provided an instrument for ultrathin, subtle manipulations. A better knowledge of the cell has cleared the way for laboratory and even industrial uses of catalysts, the cellular proteins (enzymes). In their presence chemical reactions proceed swimmingly - without heating, at normal temperature and pressure, but billions of times as vigorously. Since the action of enzymes is strictly selective and directional (each has definite compounds as the target, and its effect is strictly oriented), they evolve as precious aids for most unorthodox manipulations on live tissue.

Gene engineering owes its burgeoning to intriguing enzymes, the restrictases. In DNA they display phenom-

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enal precision in recognizing most different nucleotide sequences, the order of "code letters" and attack, cut such sequences at a preassigned point. The nomenclature of restrictases is expanding, and it now comprises more than 400. And so biologists can cut, excise and recarve DNA to desired segments and obtain individual genes, and then join ("suture") them in a new sequence.

Another enzyme, the DNA ligase, was known before the discovery of restrictases. Ligase repairs a break in a DNA molecule and restores it back to normal.

Like restrictases, ligases are an all-important tool without which a "reshuffling", or recombination, of genes would be impossible. To obtain a hybrid, recombinant molecule, it is not enough to cut it into selected segments. The main thing is to combine these encoded segments into a "coherent text", i.e. a desired experimental program. No tinkering is permissible here. That's where ligase is quite handy First an isolated DNA is treated with restrictases, and then with ligases supplemented to the solution; this way a researcher may get any combinations in vitro, in a test-tube. The interspecies barrier is an insurmountable obstacle in nature, the main stumbling-block in artificial selection. But this barrier is virtually nonexistent in genetic engineering.

Today the art of recombination involves certain pathways of inducing selective modifications: say, when foreign genes are injected into a microbial cell - these may be even human genes! Here dramatic modifications may be achieved in the hereditary program - so much so that a foreign gene will foist its type of metabolism, and the host cell will start producing what it has never done before, e.g. human insulin, a substance that can now be produced on a commercial scale. Insulin is a vital protein excreted by the pancreas and ranked in the category of hormones. It regulates the carbohydrate metabolism, specifically, the level of sugar in blood.

A shortage of this hormone causes diabetes, a grave disease which is no respecter of persons.

Here gene engineering provides an effective way out, in combination with chemical methods of synthesizing nucleotides, the monomeric units of nucleic acids; these methods have been developed by the American biochemist and Nobel prize-winner H. Khorana and other scientists. The Soviet Institute of Bioorganic Chemistry named after M. M. Shemyakin has just completed its work on a preparation of human insulin produced with the aid of bacteria. First, short synthetic gene fragments are joined by ligase. The gene thus obtained - it programs the "assembly" of an insulin protein - is joined to a recombinant DNA supplemented with bacterial or yeast cell organelles. This combination of the chemicoenzymic method with gene engineering techniques enables us to obtain the primary product, the pro-insulin, produced by bacteria. Pro-insulin can be readily converted to insulin, and at low cost at that. By the way, native human insulin is formed via the pro-insulin stage. This is one of the ocular examples of commercial biotechnological production.

Even though the practical results are quite impressive, this problem is far from the ultimate solution.

Early in the 1950s D. Ladeberg, working on the colibacillum E.coli, which is a continuous satellite of the human intestine, discovered - apart from the main DNA, always present in a host cell - also small autonomous DNAs which bacterial cells readily exchanged. Then it came out that higher organisms, too, have small DNAs , the plasmids, in their cell cytoplasm; and this is besides the principal, nuclear DNA.

Plasmids drew attention from medics in the first place. Trying to find out why antibiotics, so much effective against dysentery, fail in some patients, they detected this phenomenon: infectious bacteria had plasmids containing several genes resistant to antibiotics. Then came another insight: all genes resistant to antibiotics (quite a headache for clinicians battling against all the various infections) are always present in plasmids. Since the autonomous DNAs are readily exchanged by bacteria, they develop resistance to antibiotics pretty fast. For instance, the staphylococcal infection (staphylococcosis), a real plague for surgical clinics, was diehard just because of the plasmids. That is why the latter became an object of close study.

Yet this curse for practicing doctors-the ability of plasmids to "change hands" and move from bacteria to bacteria-proved a real windfall for genetic engineering. If plasmids isolated from bacteria are inoculated a foreign DNA and then such hybrid plasmids are "implanted" in bacterial cells, part of the hybrids is bound to be viable and will be proliferating in bacteria.

Using restrictases and ligases microbiologists design hybrid structures containing DNA fragments from any organisms. Thereupon such hybrid plasmids are bred in host bacteria that reproduce the inserted fragment of DNA many times over. This is cloning. It is cloning that gives a thrilling opportunity of enhancing the capability of live cells to synthesize and accumulate new substances in large amounts. Such is the basic principle of gene engineering.

The M. M. Shemyakin Institute of Bioorganic Chemistry is on the leading edge of such research in this country This center has carried out fundamental studies on the biosynthesis of human interferon. This remedy inhibits proliferation of viral DNA and builds up immunity in sound cells to viral infection. However, a large amount of interferon is needed for clinical tests alone, let alone for full-scale medication. Just one milligram of interferon can be extracted from a liter of blood - for one sole injection only. So you see how vital it is to search for alternative sources of this precious remedy One such source is available today - microorganisms designed by gene engineering techniques and capable of high- performance synthesis of human interferon. A mixture of messenger RNAs coding for various proteins contains just - 0.1 percent of RNA-encoding interferon. Nonetheless we have managed to isolate genes of three interfe-

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rons - human leucocyte, fibroblast and immunity interferons - and introduce them into E.coli plasmids and clone them. This accomplishment is tantamount to a revolution in basic and in applied research alike. Thanks to the achievements of research staffs at the Institute of Bioorganic Chemistry and the All-Union Research Institute of Genetics it has become possible to launch the production ofinterferons on the basis of E.coli strains. We have "tamed" bacteria capable of synthesizing hundreds of micrograms of this valuable substance per liter of a cell-containing solution.

The ever-expanding arsenal of gene engineering makes for ever better techniques of interferon production. Bacterial genes have now been cloned in yeast and in cells of higher animals having a cell nucleus. This method promises to become a most effective basis of commercial production for other human interferons in the near future. Furthermore, comparative studies of different interferons and their structural and pharmacological properties have prepared the ground for selective modification of genes with the aim of improving their useful properties. Hybrid genes are being designed that will program synthesis of hybrid interferons in a host cell. Researchers of the Institute of Bioorganic Chemistry have designed hybrids designated alpha-A, alpha-B and alpha-C that are significantly different in their characteristics from the stock material. Progressing apace, genetic engineering methods allow to "implant" human genes in bacterial cells and design strains of microorganisms (like E.coli, grass bacillus, yeast, etc.) capable of synthesizing essential hormones; such methods also allow to obtain synthetic blood proteins in no way different from native human proteins. All things considered, medicine is heading for a real revolution. Physiologically active substances will give rise to a new generation of medications obtained from human proteins. Such remedies are strictly targeted in their action and cause no aftereffects.

Here's one graphic example. Research workers of the Laboratory of Functional Enzymology (Institute of Molecular Biology, USSR Academy of Sciences) have obtained somatropin, the human growth hormone, in an E.coli culture. How could they do that, making the human gene work in a microorganism, in a foreign medium, just as effectively as in the host organism?

The outline of this technique: E.coli regulatory elements, essential for the synthesis of RNA and protein, were "stuck" to the human gene. That is, the human gene was converted to a functional, bacterial gene controlling the production of the growth hormone to the desired "human program". Since today we have copious evidence on regulatory gene- activating sequences, it became possible to Select the most effective combination of such sequences. This "clever-clever" design, once transferred to the microorganism's cell, gave rise to a bacterial culture capable of synthesizing the required product in large amounts: hundreds and hundreds of molecules per cell.

The objective of all this research is not to demonstrate the possibilities of gene engineering-it has practical

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implications, the production of the growth hormone. The next stage, just as important and difficult, relates to the sphere of biotechnology as such: namely, optimizing the cultivation of bacterial colonies and purification of the end product. In our country such kind of R&D work has been carried out by the Academy's Institutes of Molecular Biology and Bioorganic Chemistry in cooperation with research centers of the Ministry for the Medical Industry and GLAVMIKROBOPROM, a body charged with the production of microbial cultures. Pooling their efforts, research staffs have developed an experimental medicinal preparation now being tested for subsequent clinical trials.

The human growth hormone, composed of 191 amino acids and interferon (its "alpha" brand comprises 166 amino acids) belong to rather large proteins. But many physiologically active substances, such as hormones and neuropeptides, happen to be much smaller in size. If we take E.coli, it can hardly produce short peptides of 30 to 40 amino acids or very long ones. Too small sequences might be just digested in bacterial cells. In this case microbiologists resorted to a device they learned from Mother Nature: develop hybrid proteins where one of the components is taken from some common protein that could be readily synthesized in a microbial cell; and then join in a required protein by an amino-acid connective.

The Institute of Molecular Biology has synthesized another essential human hormone, calcitonin, excreted by the thyroid gland. The gene is assembled of small fragments and joined by ligases.

Ever new genes, responsible for the production of human and animal proteins, are isolated. These are enzymes dissolving blood clots, and these are new hormones and substances regulating hormonal metabolism.

It is worth to mention the work to modify the hereditary program of plants, in particular, methods of molecular hybridization. Taken as stock material in this case are not sex cells (gametes) but somatic cells (somas), i.e. those of the body of plants. These cells are treated with special enzymes to remove the hard shells; the cells thus stripped - isolated protoplasts - are hybridized. From such hybrid cells we can cultivate a cell mass without changing the cultivation conditions and, inducing the cell colony to orderly growth, obtain hybrid plants as the end product. This molecular hybridization technique is not only a new instrument of genetic analysis, it is also a welcome addition to the practical arsenal of crop selection. This work is part of the goal-oriented program BIOTEKHNOLOGIYA (Biotechnology). Hybrid cultures of potato and tobacco have been evolved and tested by the Botany Institute of the Academy of Sciences of the Ukrainian SSR and the Plant Physiology Institute of the Academy of Sciences of the USSR. The first specimens of these plants are now tested in field conditions.

Artificial genes are a no less promising area of research. An artificial foreign gene "implanted" into a cell imposes new information on it. Ultimately this cell gives birth to a plant with preassigned characteristics. We in this country have evolved a hardy plant resistant to antibiotics by inserting the gene of an appropriate microorganism.

This pattern of cloning has been designed for key farm crops: protoplast -> cell culture -> integral plant. The liposomal techniques of inserting foreign genetic material into protoplasts are now being customized. Another area involves the DNAs of bacterial and viral plasmids, as well as the native extrachromosomal DNA of plants, for imparting hereditary information and breeding hardy strains resistant to diseases and not dependent overmuch on the soil composition and level of moisture. Self-fertilizing plants look like a great idea. Contemporary farming is inconceivable without a proper supply of nitrogen fertilizer. To produce it mankind is expending more than 1.5 percent of the available energy and unrenewable fuel supplies like oil. The utilization efficiency of nitrogen fertilizer is 50 percent at best. That is to say, of the 50 mln tons of nitrogen fertilizer produced in the world as much as 24 mln tons decomposes in top soil into noxious oxides that poison the environment. But the biological fixation of nitrogen is fueled by the energy of the sun; fixed by bacteria, such nitrogen is assimilated fully. On cultivated lands biological nitrogen is provided by nodule bacteria, or rhizobia, and blue-green algae. So the problem confronting biochemists: look into the genetics of nitrogen fixing nodule bacteria and master the molecular-genetic mechanism of nitrogen fixation.

This is not to mean that gene engineering and new biotechnologies are a nostrum, a cure-all remedy destined to supplant conventional methods. However, new molecular- biological approaches can be applied with much success for improving the protein composition, for protection against diseases and environmental pollution. That's an obvious fact today. It's often hard to predict important spin-offs from basic research and their unexpected practical uses.

Needless to say, gene control skills are an awesome responsibility and a risk factor. But on the other hand, we had better acquire such skills rather than let genes control us. Certain safeguards are a must. We must be ready to counter all possible risks and hazards. The Committee on Genetic Experimenting is closely involved with the work of elaborating standard rules and safeguards for genetic engineering. Here in the Soviet Union bioengineering research proceeds in full conformity with the international safety standards for recombinant DNAs. Conservative caution and a high sense of responsibility are always best: now, in the nuclear age, and in the forthcoming genetic age too.

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