Physiological Genetics: The Genetic Code and Engineering | Botany

In the below mentioned article, we will discuss about physiological genetics. After reading this article you will learn about: 1. The Genetic Code and 2. Genetic Engineering.

The Genetic Code:

When the existence of the genes is agreed, the still greater problem is confronted— How are the different characters actually caused by the genes? Experiments and speculations as to this are of recent origin and come under the field of Physiological Gene­tics. It is possible that the genes, though insignificant particles, start certain physicochemical reactions in the living body which cause the develop­ment of the characters.

That some chemicals come out of the nucleus (i.e., the genes, according to the gene theory) is beautifully illustrated by the siphonaceous alga, Acetabularia. Acetabularia is a large but uni­cellular alga. The single cell is differentiated into a holdfast, a stipe and a mushroom-like cap (Fig. 843).

If the unicellular plant be cut into bits, the bits survive for a good period of time and, also, one bit from one plant may be grafted to another bit from another. It is found that if a bit contains the nucleus, which is located in the holdfast; it not only survives but also regenerates the other parts. A bit without a nucleus cannot do so. Two species of this alga, A. mediterranea and A. mediterranea be grafted on a holdfast of A. wettsteinii. A. mediterranea be grafted on a holdfast of A. wettsteinii with a nucleus, it is found that a cap develops on the mediterranea stipe but this is not of the mediterranea type but of the wettsteinii type evidently because the chemicals controlling growth are coming from the wettsteinii nucleus.

Another very recent spectacular experiment also demonstrates the role of the nucleus in heredity. In recent days great successes have been achieved in the asexual reproduction achieved from the culture of single cells of higher plants and animals in artificial media resulting in the generation of complete plants out of single cells.

In such an experiment Dr. J. B. Gurdon of Oxford University destroyed by radiation the nuc­leus of an unfertilised egg cell of an African clawed frog and transplanted within this cell a nucleus from a body cell of another frog. The new cell combination began divi­ding and regenerated a complete tadpole which matured into a frog which was nothing but an identical twin of the frog which donated the nucleus and not the frog which originally donated the egg!

Light is thrown on gene action by the study of phenocopies. It has been found that there are various races among butterflies showing different types of designs on wings. Some of these natural races may be produced in the laboratory by treating a different type by different ranges of temperature during growth. Drosophila melanogaster normally shows a light brown body. In 1910 Morgan obtained a yellow-bodied mutant.

In 1939 Rapopport found that when larvae of normal flies are fed on food containing silver salts, yellow-bodied Drosophilas may be raised. Such an artificially raised butterfly or Drosophila fly is termed a phenocopy and is not the same as the original race or mutant as these copied characters are not hereditary. Actually the artificial treatments had the same effects as the genes which cause these characters. While the genes pass on from generation to generation these artificial treatments do not.

Gene action is sometimes affected by some unknown cause so that, although present, it may show no action on the individual. When a gene shows its full action it is said to have complete penetrance. When it fails to act completely in some cases it shows incomplete penetrance. Persons inheriting genes of some serious disease may not be actually affected by the disease because the gene does not show penetrance. The expression of a gene in the phenotype, i.e., the actual appe­arance of the organism is its expressivity; reduced penetrance will cause reduced expre­ssivity.

That genes show different degrees of penetrance and expressivity does not go against the gene theory. The gene action is caused by complicated, chemical reactions resulting in the control of enzyme synthesis. There may be some other factor or even some other gene which hinders some phase of this reaction causing an incomplete penetrance.

In recent years there has been phenomenal progress in the field of biochemical genetics involving the synthesis of DNA and RNA, which has thrown much light on the action of the genes. Three awards of Nobel Prizes have already made on this subject. First, in 1959, S. Ochoa (New York) and A. Kornberg (Washington) were awarded a Nobel Prize for the synthesis of nucleic acid.

Then, in 1962, Crick, Williams and Watson got the award. Finally, in 1968, the award has gone to M.W. Nirenberg, R. Holley and Har Gobind Khorana (Fig. 844) for the elucidation of the genetic code. Khorana is now well known in India for he is an Indian who has settled in the U.S.A.

The conclusions that arise from their extensive work is as follows:

The genes control the synthesis of enzymes and other proteins which again control the development of characters. It has been seen that in a polynucleotide chain the bases are arranged in different orders. Three such consecutive bases (say AAC—i.e., Adenine —Adenine—Cytosine) forms a nucleotide triplet which is the unit for the synthesis of a particular amino acid. This unit is named codon as the unit has got a code on it which causes the particular amino acid to be synthesised.

Thus, a number of codons arranged in a particular order will cause the synthesis of the particular amino acids in a parti­cular order. These amino acids will get linked in that particular order forming a polypeptide chain which forms a molecule of the particular protein. But, protein is synthesised in particular places called ribosomes which are ultramicroscopic bodies in the cytoplasm.

So, what actually happens is that of the two polynucleotide DNA chains in a double helix gene (Fig. 845-1) one procreates an RNA replica (Fig. 845-2) which is called the Messenger RNA. This may be termed as the transcription of the message or the code on the RNA. This RNA contains the nucleotides in the same order only the sugar is changed from deoxyribose to ribose by oxidation the bases are the same excepting that Thymine (T) is changed to Uracil (U).

The messenger RNA now moves out of the nucleus into the cytoplasm and gets attached to a ribosome (Fig. 845-3) which is to manufacture the protein as coded on this messenger RNA. Even this is not done directly. Each codon on the Messenger RNA produces soluble Transfer RNA (Fig. 845-4). Usually, in RNA, there is a single helix. But, in the transfer RNA the single thread is folded over forming a double helix of which one end is closed and the other end is open.

The transfer RNA holds the codon which is the opposite (C for G, U in place of T in DNA for A or vice versa as explained earlier) while in the open end it draws out the specific amino acid fixed for that codon. This amino acid is fixed in that place. The codons on the sides also draw out the respective amino acids. All these amino acids now get linked up in their positions synthesising the exact protein molecule as already coded. This action of the transfer RNA may be described as the trans­lation of the coded message into protein.

The transcription and translation throughout the length of the messenger RNA do not proceed all at once but different codons are translated at different speeds. This control is called modulation and may be responsible for differentiation of organis. It is thus seen that the codons or nucleotide triplets may cause the synthesis only of the particular amino acids in that particular order causing the synthesis of only the par­ticular protein molecules.

Thus, the very polynucleotide chain of DNA or the gene or the consequent messenger RNA has got coded on it what protein it can manufacture. This is called the genetic code. Extensive researches have now shown which codon may manufacture which amino acid as shown in the following table for 20 amino acids.

Khorana and his students now have even succeeded in creating artificially a yeast gene of 77 nucleotides. This is, of course, the first step, as a complete chromosome contains millions of nucleotides—each single human cell has been calculated to contain chains involving some 6000 million nucleotides. Later (1976), Khorana with his asso­ciates has been able to synthesise the tyrosol suppressor tRNA gene of Escherichia coli complete with its control elements. This gene was able to function inside the bac­terial cell when inserted. This is a spectacular achievement and leads us on to Genetic Engineering.

Genetic Engineering:

The experiments of Khorana and others gave rise to wild hopes and ‘dreams’. If it be possible to synthesise genes and to introduce them into bacteria, is it not possible to create new genes and introduce them into the cells of other animals giving rise to unknown characters? It may be possible to create new antibiotics and medicines, to cure incurable diseases or even to create a ‘god’! But, with unknown factors, it may also work the other way. Instead of beneficial, dangerous and uncontrollable orga­nisms (say, those causing epidemics or even a Frankenstern) may arise.

The idea may be the source of hair-raising science fictions but, in actuality, may not be palatable at all. Since the ’70s of this century much experimentation has been done and some very hopeful results have been obtained. A new science called Genetic Engineering has developed. The aim of this science is to introduce foreign DNA into living cells by special techniques and thereby changing the gene complex and the behaviour of these organisms.

The scientists-are aware of the dangers, they have been warned and all precautions are taken so that no evil may result. In the U.S.A., where the most work of this type is being done, the National Institute of Health has formulated certain guide lines for safety and these are followed in all laboratories.

It is easy to understand that synthesising all genes is not an easy job. Secondly, it is much safer to work with known genes already present in organisms without trying to ‘create’ new genes. There is enough scope to achieve remarkable success this way. The method of genetic engineering that has been most widely used is what is known as the Recombinant DNA Method.

Recombinant DNA Method:

In understanding the Recombinant DNA Method one says of the tools required for this type of engineering. But actually no mechanical tool is meant as this is not a case of mechanical repair of a machine or that of a surgical operation with the doctor’s kit. Actually, these are cytochemical techniques which are performed in vitro.

Certainly, the methods are not easy but the general principles may be explained as follows:

(1) The gene that has to be transferred is to be isolated. This gene is called the passenger DNA as this is a gene foreign to the receiving organism and has to be transferred passively to a strand of DNA of the recipient.

(2) The passenger DNA is now cloned to the vector (also called vehicle) DNA which has been very suitably found in the plasmids of bacteria or even in bac­teriophages (Fig. 846). In bacteria, extra chromosomal DNA occurs in the cytoplasm as small circular rings. They can exist independently of the main genome set of the bacteria and multiply independently. These are called the plasmids. Being very small, they can be isolated and transferred out of the bacterium.

Alter, reintroducing the ‘recombined’ or the ‘hybrid’ plasmid into the bacterium it becomes a part of it and goes on dividing and multiplying the new characters normally.

The recombination needs very delicate manipulation which cannot be done by any apparatus but is done by biochemical techniques. Special enzymes, which have also been isolated from bacteria—endonucleases, endonucleases and, above all, the restriction endonucleases. All these enzymes are capable of cleaving by digestion a DNA (a single or paired double chain) into bits.

Exonucleases can digest or break the base of any single chain of DNA. Endonucleases cleave the DNA at any point of a DNA chain except the ends. But, the restriction endonucleases cause breaks, in particular points of nucleotide sequence of both the strands of a pair. Since this ‘particular point’ is not in the same place in the two chains, the cut becomes uneven so that the broken ends form some­thing like a mortise cut and become ‘sticky’ (Fig. 847). These sticky ends make it easy for the subsequent splicing into the vector plastid chain.

The passenger DNA or the gene to be implanted (or cloned) may be (1) a synthetic gene as first synthesised by Khorana or (ii) a complementary DNA (cDNA) which is synthesised on an RNA template with the help of reverse transcriptase on the DNA-RNA complex and is then sub­jected to alkaline digestion to isolate single-stranded .DNA; the complementary DNA is actually synthesised on this single stranded DNA by DNA polymerases.

The cDNA so produced is specific for a gene and can be linked to a vector DNA for transfer. If none of these techniques work then a gene may be isolated by (iii) a ‘shot gun’ experiment. In this method, the whole DNA of the organism from which a desirable gene is to be isolated is broken up into fragments by restriction endonucleases. Each fragment is now recombined with suitable vector DNA and each recombination tested for the required gene after transfer to a host.

Experiments following these lines have not only given deeper access to the organi­sation of chromosomes and the working of the genes but have also given us some very useful practical products and there are possibilities of more benefits.

Some of the fields where they have been applied are:

(i) Synthesis of polypeptides and proteins of medical importance, specially pro­duction of insulin and other medically important hormones, proteins and peptides.

(ii) Production of vaccines.

(iii) Production of interferon’s and specific antibodies.

(iv) Production of new strains of organisms with beneficial properties.

(v) Determination of defects and replacement therapy for genetic diseases.

(iv) Digestion of wastes, specially removal of hydrocarbon waste and its conver­sion into protein.

Some of the outstanding achievements are being mentioned below:

(1) Insulin production (Fig. 848):

The protein, insulin is secreted from the pancreas for normal sugar metabolism. The absence of this secretion causes the very common disease diabetes mellitus. It may be treated by injection of insulin which is normally obtained only from animal sources. But this source is extremely limited and insufficient Hence, this attempt at genetic engineering. If the gene which causes insulin secretion could be introduced within the patient, it would have been the ideal treatment.

The second best way has been found by transferring an insulin gene into a bacterium which would grow very, fast and would be a cheap source of insulin.

The bacterium Escherichia coli has proved to be a gold mine for gene transfer. The strain E. coli X 1776 was isolated by Curtiss in 1976 (U.S. Bicentennial year—named 1776 for this). This strain was found to be perfectly harmless and was certified by the N.l.H. for use in this type of experiments. The actual transfer was made by Ullrich and associates in 1977 in the University of California, San Fransisco campus (Bio­chemistry & Biophysics Division).

The steps followed are as follows (See Fig. 848):

(i) Messenger RNA (mRNA) was isolated from rat pancreas and complementary DNA (cDNA) was synthesised with the help of the enzyme reverse trans­criptase. A restriction endonuclease was now used which cut the DNA strands between the 3′ and 5′ nucleotides (also see Fig. 847) leaving sticky ends. E. coli X 1776 bacteria were treated with a detergent in a test tube which dissolved the outer membrane of the cells exposing the DNA strands. The plasmid rings are now separated by a centrifuge and then treated with the same restriction endonuclease so that the ring is broken at the same points and a part cut off. (The ring may now become linear.)

(ii) The open rings of the plastid DNA (which may be linear) are new placed together with the broken mice insulin DNA in the presence of the enzyme T4 DNA ligase. This results in the recombination of the broken plasmid DNA ring when the rat insulin DNA gets attached like a chimaera.

(iii) The recombinant DNA is placed in a solution of cold calcium chloride con­taining normal E. coli X 1776 cells. This solution is suddenly heated so that the bacterial outer membrane becomes permeable allowing entrance to the hybrid recombined plastids.

The recombined plasmid mw divides along with the bacteria duplicating the genes as in normal cases.

The experiments have been simply described but they are by no means that simple. There are possibilities of footfall at every stage. Actually, Ullrich et al did not show that the new cloned bacteria do secrete insulin. This was shown next year (1978) by Gilbert (who performed the cloning by a different technique) who found that minute quantities of proinsulin (precursor of biologically active insulin) were secreted by the recombined plasmids. But, still, this technique needs lot of improvement to be of practical use.

(2) Transfer of nitrogen fixation:

Certain bacteria fix atmospheric nitrogen by their nif genes. It has been found possible to transfer this nif from nitrogen-fixer to non-fixer bacteria. The aim now is to transfer the nif gene to the crop plants themselves—first to the symbiotic legume plants and then to crop plants like rice or wheat when all such crop plants would be able to utilise atmospheric nitrogen without the help of bacteria. Such a goal does not seem beyond reach today.

(3) It has been found possible to transfer the human hormone hGH (an amino acid protein) to E. coli. This gives rise to great hopes in the field medicine and pharma­cognosy.

(4) A spectacular genetic engineering success has been achieved by Dr. A.M. Chakravarty of Calcutta who now works in the General Electric Research Laboratory in the U.S.A. He has developed by genetic engineering a new type of the bacterium Pseudomonas which can gulp hydrocarbons. This is to be used for clearing accidental petro­leum oil spills which sometimes spread over hundreds of kilometres of ocean surface and becomes a serious danger to the littoral countries. So important is this discovery that the U. S. Supreme Court has ruled that a patent should be granted for this bac­terium—for the first time for the ‘manufacture’ of any living organism.

(5) Of late, a substance called interferon (IFN) has been discovered as a soluble substance secreted by cells infected by virus. This substance is now known to be a group of small proteins. It can also be produced by sensitised lymphocytes and may be induced experimentally in cells by treating them with double stranded RNA. It has been found that this substance has immense possibility in the immunisation and treat­ment of various hitherto incurable virus diseases and even cancer. But the substance is obtainable in so little quantity that it is almost impossible to use it commonly.

Now (in the 1980’s) it has been found possible to produce it more easily by genetic engi­neering. The interferon mRNA has been isolated from leucocytes, converted into cDNA by reverse transcriptase, cloned to E. coli and this has been shown to be active in vitro as well as in vivo. So, it may be possible to produce interferon in greater amount in future. By 1982 interferon is being used on patients in cancer hospitals. But, some fatal results have already stopped its use in French Government programmes.

(6) Recently (1982), closed DNA molecules containing genes causing secretion of enzymes have been injected into the eggs of mice and such genes have after been found to be transmitted to the off-springs. But, is most cases it is doubtful if the transferred genes have at all been able to generate the same enzymes. These are pro­blems to be solved in future.