Microbial Variations [Genetic Manipulation in Microorganisms]

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Chapter: Pharmaceutical Microbiology : Microbial Genetics and Variations

Importantly, the very fundamental unit of biological relatedness prevailing predominantly in various species as well as in bacteria that reproduce sexually are invariably defined by the prevelent ability of its members to copulate with one another.



Importantly, the very fundamental unit of biological relatedness prevailing predominantly in various species as well as in bacteria that reproduce sexually are invariably defined by the prevelent ability of its members to copulate with one another. In this manner, the species do retain their ‘basic identity’ articulately by virtue of the fact that there exist certain natural barriers that particularly check and prevent the ensuing genetic material existing between the unrelated-organisms. Ulti-mately, this critical identity is retained overwhelmingly via one generation to another (i.e., sustaining the so called ‘heredity’).


It has been well established that such organisms which reproduce asexually the basic concept of a species solely rests upon the nature’s capability to check and prevent the exchange of the ‘genetic material’ occurring amongst the ‘unrelated members’. One may, however, come across the above phenomenon quite abundantly amongst the microorganisms even though they occupy the same kind of habitat.


Example : E. coli and Clostridium spp. : In fact, these two altogether divergent organisms usually found in the ‘animal gut’, but these are quite unrelated. Furthermore, they fail to exchange the ensuing ‘genetic information’, and thus enables the proper maintenance of these species very much in an absolutely common environment. In fact, the entero-bacteria predominently exhibit such vital restrictions that could be seen amongst these types of closely related organisms.


Biologically Functional DNA Molecules : The meticuolously designed tailor-made biologically functional DNA molecules in the test-tube (i.e., in vitro) could be plausible and feasible based upon the enough concrete evidences pieced together with regard to the knowledge of the ‘nature of genetic material present in the living systems’. In other words, one would safely conclude that the construction of DNA might not only replicate faithfully, but also maintain its originality gracefully.


Chang et al. (1973) made an epoch making discovery of constructing a miraculous biologically functional DNA molecule in a test tube which explicitely combined genetic information from two different sources.


Methodology : The design and construction of such hybrid molecules were duly accom-plished by carefully splicing together the ‘segments’ of two altogether different plasmids, and subse-quently, inserting this composite DNA plasmid strategically right into the pervailing E. coli cells. At this location, it replicated duly and thereby succeeded in expressing the information of both parental plasmids.


By adopting the identical procedural details the ribosomal genes of the toad Xenopus were strategically introduced into the E. coli wherein these organisms not only replicated effectively but also expressed genuinely. Nevertheless, the RNA-DNA hybridization technique duly detected the expression of the inducted genes. Thus, the newly formed ‘DNA composite molecules’ were termed as DNA chimeras. These may be regarded as the molecular counterparts of the ‘hybrid plant chimeras’ that can also be generated by ‘grafting’*. During the past couple of decades an enormous copius volume of researches have been duly performed rather on a fast-track, and eventually this new kind of work is termed as ‘plasmid engineering’ or more recent terminology ‘genetic engineering’.


Various Steps Involved in Gene Manipulation and Selection :


There are in all four cardinal steps that are intimately involved in accomplishing the most widely accepted and recognized procedure of the gene manipulation and selection, such as :


(1) Method for cleavage and joining DNA molecules from different sources,


(2) Search for an appropriate ‘gene carrier’ which may replicate itself as well as the ‘foreign DNA’ attached to it,


(3) Method for introducing the composite DNA molecule into a bacterial cell, and


(4) Method for strategical selection for ‘clone of recipient cells’ from a rather huge population.


Discovery of Ligases :


Ligases usually refer to — ‘the class of enzymes that catalyze the joining of the ends of two chains of DNA’.


Khorana et al. (1970) first and foremost discovered that the ligase specifically produced by the bacteriophage T4 might occasionally capable of catalyzing an end-to-end attachment of an absolutely separated double stranded DNA segment only if the ‘respectively ends’ of the two segments are able to recognize each other duly.


Even though the above mentioned procedure happens to be not so rapid and efficient, but it definitely paved the way for ‘intelligent joining’ of the DNA molecules.


Salient Features :

The salient features of the genetic manipulation are as given below :


(1) DNA terminals (ends) of certain bacterial viruses may be joined together by the phenom-enon of ‘base-pairing’ existing between the complementary sequences of such ‘nucleotides’ that are essentially present on the single strand segment projecting from the ends of these molecules.


(2) Synthesis of longer segments of DNA could be achieved by adopting the principle of link-ing together the DNA molecules by means of the single strand projections using wisdom, knowledge, and skill.


(3) Terminal transferase, a relatively a recent and new enzyme, was discovered miraculously that exhibited the much desired ability to add strategically the nucleotides at the 3-end of DNA. In fact, this remarkable scientific gain of knowledge widely opened the flood-gate to-wards the meticulous construction of a plethora of highly specific DNA segments having critically the ‘single strand nucleotide molecules’ ; and, therefore, providing a potential avenue for joining the two pieces of DNA.


Example :

To link the DNA of animal virus SV40 with the bacterial virus DNA :


Figure : 6.13 illustrates the various steps that are involved sequentially to explain the construction of the recombinant DNA.


(1) First the circular DNA molecule undergoes cleavage to yield two linear DNA molecules.


(2) Under the influence of the enzyme ‘exonuclease’ the two fragmented linear DNA molecules give rise to terminally attached newer elongated segments of DNA.


(3) Terminal transferase helps these two segments of DNA to enable them hook on further addi-tions with respective amino acids (viz., A and T).


(4) Annealing process comes into being that specifically helps the two loose ends of the modified linear segments of DNA molecules to come closure in the form of a ring (not a close ring).


(5) Presence of exonuclease III and the DNA polymerase do help forming a circular modified DNA molecule.


(6) Finally, the DNA ligase renders the resulting product into a well-defined new desired ‘Recombinant DNA Molecule’.


Generalized Procedure for Constructing Recombinant DNA Molecule and Cloning :

It has been duly observed that the ‘biologically active DNA’ predominantly occurs as explicitely distinct covalently-closed circles (CCC). Nevertheless, it is first and foremost absolutely necessary to afford cleavage of ‘circular DMA molecules' to give rise to the formation of ‘linear DNA molecules’ having essentially free ends.


EcoRl, and endonuclease, is observed to be an excellent enzyme most appropriate for opening up the closed circular DNA molecules speedily and efficaciously. Besides, it possesses the superb capability to cause an effective cleavage of DNA at the specific sites exclusively. Subsequently, the resulting cleaved linear DNA molecules are then duly treated with an enzyme exonuclease, which in turn predominantly ‘chewed back' the two 5Z ends of the DNA molecules thereby allowing the single-strand- ends projecting outwards prominently. It is apparently followed up by the aid of the enzyme terminal transferase, whereby a block of ‘adenines’ got hooked on to the 3Z end of one of the two DNA species and a block of thyamines is critically attached on to the 3Z end of the other species. Ultimately, these rw species on being mixed carefully do allow the ‘quick recognition' of the complementary blocks strategi¬cally located at the two ends, get lined-up (i.e., alligned), and produce the much desired ‘hybrid mol¬ecules'. At the final stage the molecules thus obtained are adequately sealed with DNA ligases.


Figure : 6.14, depicts vividly the generalized method which is invariably adopted for duly con-structing the recombinant DNA as well as cloning.


The following steps summarizes the various sequential modes for obtaining the chimeric plasmid, the transformed cell, and the daughter cells.


(a) Plasmid gets cleaved to corresponding linear molecules by endonuclease ; also accom plished by ‘foreign DNA’.


(b) Annealing process commences to obtain the desired closed circular DNA.


(c) ‘Chimeric plasmid’ is duly accomplished via ‘ligation’ with DNA ligase.


(d) Transformed cell is obtained subsequently due to the transformation of organisms.


(e) Daughter cells are ultimately obtained from the respective transformed cell.


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