Gene regulation

Meaning of Gеnе Regulation. There is experimental evidence to show that the genes do not operate to produce enzymes all the time. Each gеnе is normally more or less repressed (inactive); but, when а particular enzyme is needed, the геlevant gene becomes depressed (active) to increase the production of that enzyme. А fully depressed gene can produce а fantastically large amount of enzyme. If the synthesis of all the enzymes is fully accelerated, metabolic chaos would ensue in the cell. Thus, the phenomena of gene repression and depression provide а means of decreasing or increasing the rаtе of synthesis of а particular enzyme in response to variations in the environmental requirement for the enzyme. In other words, а cell has а mechanism that controls the expression of а particular gene and thereby the amount of а particular enzyme at а particular time. The rate of protein synthesis is regulated by genetic apparatus and environmental factors in accordance with the need of а cell.

Meaning of Genetic Engineering

Genetic engineering is the manipulation of genes by mаn. It refers to artificial synthesis, modification, removal, addition and repair of the genetic material (DNA) to alter the phenotype at will. It has evoked а great interest because it may some day enable the geneticists to set right the disease-causing genes for the improvement of human rасе, and even to create lifе.

Techniques of Genetic Engineering

Old Technique. Breeding (hybridization) of plants and animals to change the genotype and phenotype of thе hybrids in the oldest and the most widely used technique of genetic engineering.

New Technique. Now new techniques have been developed to manipulate the genetic material. These include recombinant DNA technology, transformation and transduction.

Recombinant DNA Technology

It is а recent biological science that started in late seventies of this century. It involves the formation of recombinant DNA and its introduction into an appropriate host. Recombinant DNA is the DNA formed by combining DNAs from different organisms. This requires а skilful handling of the genetic material and, therefore, the term genetic engineering is used for it. The properties of DNA which help in the formation of recombinant DNA are denaturation and renaturation. Denaturation is the separation of the two strands of DNA by breakdown of hydrogen bonds on heating. Renaturation is the reunion of complementary strands to form DNA duplex on cooling. Thus, the single strands of DNA from different sources can join if there are segments having complementary base pairs.

It is advisable to have а brief knowledge of the tools used in the recombinant DNA technology before going into the synthesis of recombinant DNA.

А. Tools. The biological tools' used in the synthesis of recombinant DNA include enzymes, vehicle or vector DNA and passenger DNA.

Enzymes. Many kinds of specific enzymes аге employed in genetic engineering. These include lysing enzymes, cleaving enzymes, synthesizing enzymes, joining enzymes, and alkaline phosphatases.

Lysing Enzymes. These are used to open up the cells to get DNA for genetic experiments. Lysozyme is commonly used го dissolve the bacterial cell wall.

Cleaving Enzymes. These are used го break DNA molecule. They are further of 3 kinds exonucleases, which cut off nucleotides from 5' or 3' ends of DNA molecule, endonucleases, which cleave DNA duplex at any point except the ends, and restriction endonucleases, which cleave DNA duplex at specific points in such а way that single- stranded free ends project from each fragment of DNA duplex. These single-stranded free ends are called "sticky ends" because they can join similar complementary ends о1 DNA fragment from some other source. Restriction endonuclease was found by Arber in 1962 in bacteria. Restriction endonuclease-1 (ECOR— 1) is found in the colon bacterium Escherichia coli. It recognizes the base sequence GAAT7C in DNA duplex and cleaves its strands between G and А as shown below. The arrows indicate the sites where the enzyme cleaves the sequence.

3’ – CTTAAG – 5’

5’ – GAATTC – 3’

Synthesizing Enzymes. These play а role in the synthesis of DNA strands on suitable templates. They are further of 2 types: Reverse transcriptases, which help in the synthesis of complementary DNA strands on RNA templates; DNA polymerases, which aid in the synthesis of complementary DNA strands on DNA templates.

Joining Enzymes. These help in sealing gaps in DNA fragments which are otherwise joined by complementary base pairing. Tu ligases are examples.

Alkaline Phosphatases. These cut off phosphate group from the 5’ end of linearised circular DNA tо check its recircularization.

Vehicle DNA. The DNA used as а carrier for transferring а fragment of foreign DNA into а suitable host is called vehicle DNA. Two types of DNA are used as vehicles: plasmid DNA and bacteriophage DNA.

Plasmids. The plasmids, already described in chapter 2 of this unit, are small, circular DNA molecules present in bacteria in addition to the chromosomal DNA. They may pick up genes Грош one bacterium and transfer them tо another. They retain their characters after combining with the DNA of another organism. Not being а part of the main genome, they can bе easily isolated and transferred. These features make the plasmids suitable for use as а vehicle DNA.

Bacteriophage DNA. The bacteriophages, or simply phages, as mentioned earlier in this chapter, are viruses that infect and kill bacteria. Their circular DNA is also suitable for use as а vehicle DNA.

Passenger DNA. It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA. Three types of DNA are used as passengers: complementary, synthetic and random.

Complementary DNA (cDNA). It is synthesized on RNA template with the help of reverse transcriptase enzyme. The DNA strand is isolated from the hybrid RNA - DNA complex by using alkaline phosphatase enzyme. А complementary DNA strand is then synthesized on the isolated single-stranded DNA template with the help of DNA polymerase. The cDNA duplex so formed can be joined to vehicle DNA for introduction into а host cell.

Synthetic DNA (sDNA). It is synthesized with the help of DNA polymerases on DNA template or from free deoxyribonucleotides without а template.

Artificial Synthesis of DNA on Template. In 1961, Kornberg and his coworkers synthesized DNA from а mixture of deoxyribonucleoside triphosphates, DNA polymerase enzyme, metal ions and а segment of viral DNA to act as а primer. The synthetic DNA showed biological activity. Vixen introduced into bacteria, this DNA coded for the formation of virus particles similar to the one from which primer DNA was taken.

Artificial Synthesis of DNA without а Template. Hargobind Khorana and his colleagues produced the first synthetic DNA in 1965 by purely chemical means. They synthesized the gene coding for yeast alanine — tRNA which needed only 77 base pairs. Unfortunately, this gene did not function. Khorana and his associates later synthesized the gene coding for tyrosine — tRNA of Е. Coli, which functioned is the bacterium Е. соli when introduced into it in combination with а phage DNA.

Random DNA. It refers 1о small fragments formed by breaking а chromosome of an organism with the help of restriction endonucleases.

В. Formation of Recombinant DNA (Integration of Passenger DNA to Vehicle DNA). The DNA to be used as passenger or vehicle is taken out from the cell by lysing it with а suitable enzyme. The DNA is isolated from other cell contents by ultracentrifugation and purified. Then both the passenger and the vehicle DNAs are cleaved by using the same restriction endonuclease so that they have complementary sticky ends. They are mixed under suitable conditions. Their complementary sticky ends pair and their ends are sealed with ligase. This produces а recombinant DNA.

Introduction of Recombinant DNA into Host. Earlier Е. coli was used as а host for the recombinant DNA. Now Bacillus subtilis and yeast are also used. Тhе bacteria to be used as hosts should be without plasmids. Their wall is made permeable by treatment with calcium chloride or lysozyme. The recombinant DNA is added to the culture in which such bacteria are growing. The recombinant РМА is taken up by bacteria along with nutrients. It replicates when bacteria divide and may express the information of both the parent DNAs. The hybrid cells formed m the above process may bе considered new species.