The below mentioned article provides an overview on Cells and Organisms Used in Molecular Biology.
For any molecular biological work or genetic engineering, the synthesis and cloning of any recombinant DNA involves introduction of this DNA into bacteria.
Structure of E. coli:
Among bacteria, escherichia coli are suitable for this purpose for the following reasons:
i. It can be grown in a very simple and inexpensive media.
ii. It is also produced in large numbers within a very short time.
iii. It has the ability to take up a great variety of reconstructed plasmids and phases.
Another suitable bacteria is Bacillus subtilis. However, the most commonly used bacteria in all cell biological work is Escherichia coli. Many genetical and molecular biological experiments have been done with this bacterium.
Numerous genetically distinct strains of E. coli are available in many laboratories. There are about 3000 genes in the circular DNA of E. coli and the genetic map of most of the genes has been done (Fig. 5.1).
E. coli is a rod-shaped bacterium consisting of two layers of cells—inner and outer membrane. The outer cell layer consists of a protein- phospholipid region and a layer of peptidoglycan. This peptidoglycan layer surrounds the inner membrane and cytoplasm of E. coli.
There is a space between the two layers of the cell known as Periplasmic space. The inner membrane has an important role in maintaining the selective permeability of the bacterium. In other words, it controls the permeability by determining which molecules will enter or leave the cytoplasm.
The cytoplasm contains a single, double-stranded chromosome of about 1300 µm length which has been superseded to remain in the bacteria of length 3 µm and diameter of 1 µm.
E. coli can be grown in a very simple nutrient broth containing 0.5% NaCl, 0.5% yeast extract and 1.0% Bactotryptone. For selecting and propagating genetically uniform colonies of bacteria, a very dilute suspension of bacteria is grown on a solid medium resulting in the development of compact colonies originating from a single cell within 12-24 hours.
Few cells from this colony are then sub-cultured in a liquid medium to get a large mass of genetically identical cells within 12 hours at 37°C. The solutions become turbid due to heavy growth of the bacteria. The probability in spontaneously inducing mutation is about 1 in 106 times replication of each gene.
Thus, during growth of cells in culture media, detection of mutations is being done in culturing cells in some selective conditions so that cells with some important characteristics will grow. In this way strain with antibiotic resistant characteristic and some! specific nutrient requiring strains have been isolated and selected.
The mutations in bacterial culture can be assayed randomly—either through replica plating technique (Fig. 5.2) or through Patch pattern technique. The sensitivity or resistance to antibiotic or nutrients can be identified by growing the bacteria in a minimal medium containing a specific bacteria or nutrient. The normal method of plating of bacteria is shown in Fig. 5.3.
Every detectable mutation can be isolated and it can be described as a new strain. The nomenclature to designate mutations was done by Demerec et al (1966). The three letters in static in lower case are referred to as mutated locus which can be exemplified as his for the histidine gene, gal for the galactose gene etc.
The next capital letter shows the regulatory element (operator or promoter) present at that locus. Finally a number is given to describe the particular mutation. For example, his A38.
Some strains of bacteria contain phage DNA which has also been included in the genotype. As for example, nrd A his C (c 1857) shows that the bacterial strain is mutant in nrd A and his C locus and it contains phage with some mutations.
Bacterial Strains Resistant to Antibiotics:
Antibiotics are compounds that can inhibit bacterial growth at low doses and these can be bacteriocidal or bacteriostatic at high doses. But, in the bacteria, plasmids contain some genes which can counteract the action to antibiotics. In other words, these genes offer properties of resistance to antibiotics to the bacteria.
The antibiotic resistance properties vary according to the method of action of these antibiotics on the cell.
These are:
i. Ampicillin is a derivative of penicillin that kills bacteria by blocking some reaction step in cell-wall synthesis of bacteria. Resistance to Ampicillin is due to the presence of an enzyme β-lactamase that breaks the ring of β -lactam of the antibiotic.
ii. Colicin E1 kills bacteria by some modifications in the structure of the bacterial membrane. Colicin E1 resistance bacteria have a special protein (encoded by a genecea) which binds with colicin E1 to stop its action.
iii. Tetracycline kills bacteria by inhibiting the bacterial protein synthesis in associating with the 30S ribosomal sub-units. Tetracycline resistance strains having a resistance gene (tet) produces a protein which prevents the transport of the antibiotic to the cytoplasm of the cell.
iv. Chloramphenicol also kills bacteria by inhibiting bacterial protein synthesis in associating with the 50S ribosomal sub-units. Bacteria, resistant to this antibiotic, have a special enzyme, chloramphenicol acetyl- transferase, which inactivates the ‘antibiotic through acetylation.
v. Kanamycin also inhibits bacterial growth through inhibition of protein synthesis by attaching with the 70S ribosomal sub- units. It also induces erroneous reading of mRNA. The strains having resistance gene modifies the antibiotic in such a way that it cannot bind with the 70S ribosomes.
vi. Streptomycin also leads to errors in mRNA by joining with the 30S sub-unit of ribosomes. The resistance gene prevents the action of this antibiotic like Kanamycin.
Growth Phases in Bacteria:
Under suitable conditions, double the number of bacteria occurs in every 20 minutes due to their binary fission.
When a small amount of bacterial cell (inoculum) is cultured in the culture medium, 4 different growth phases (Fig. 5.4) can be distinguished:
i. The lag phase:
During this time no growth occurs, which means certain time is needed to start division which is known as the Lag phase.
ii. The exponential phase or Log phase:
In this stage, cell growth and multiplication occurs at a constant rate. The period of this phase is generally 10 min to 1 hr. at 37° C. Most of the experiments are carried out in this phase. One great advantage of this phase is that the cells can be diluted to a fresh medium without affecting the growth rate. Hence, the bacteria can be grown in this phase for an indefinite period of time.
iii. Stationary phase:
During culture, when there is a change in the media composition or the oxygen concentration becomes less, the growth of bacteria gradually comes to a stop.
iv. Death phase:
At the end of the stationary phase, the number of viable cells diminishes and finally leads to the death phase.
Growth Media:
(i) M9 media:
Dissolve in 1 liter of double distilled water.
The medium is autoclaved after adjusting the pH to 7.4. Then 2 ml of sterile 1M MgS04 and 100µl of lm CaCl2 solutions are added. Growth of the bacteria can be enhanced by supplementing with 0.2% Casamino acids, 0.2% glucose.
(ii) LB (Luria-Bertani) Broth:
These are dissolved in 1 litre of double distilled water. pH is adjusted to 7.4. 0.2% glucose can also be added to the media. For solid media 15g of Agar is added before autoclaving.
Spreading Technique for Culture:
All bacteria growing in log phase or early stationary phase can form isolated colonies on agar plates. Since the cell density in a growing culture is generally 107 cells/ml, it is important to dilute them 10 to 100 times before spreading on a plate.
The method of spreading of a bacterial culture is shown in Fig. 5.5(a):
Method of Isolation of a Single Colony:
The isolation of a single colony in case of bacteria is essential to avoid contamination and the possibilities of heterogeneity. This is possible by culturing the cells in an agar plate. When the colony is about 2 mm in diameter, a single colony is picked by the flame-sterilised loop.
It is then transferred to a sterile tube containing 1 ml of growth medium and cultured overnight in a constant temperature water bath. Then a primary streak of the inoculum is made on a fresh plate by dipping the sterile loop in the culture tube. Other streaks are made with the help of this loop as shown in the Fig. 5.5(b).
Bacterial cultures can be stored for a few weeks on petridishes at 4°C after sealing with parafilm. Using Glycerol (15%) as cryoprotectant, bacterial cultures can be stored in the cryogenic tubes at -70°C to -80°C.
The idea of transfer or exchange of genes came from the study of the three processes of bacteria in the transfer of genetic material. The genetic processes in bacteria are different from eukaryotes. Here, cells are always haploid and there is no true zygote.
These 3 processes are:
(1) Transformation,
(2) Conjugation and
(3) Transduction.
i. Transformation:
It is the genetic change of a bacterium due to the presence of isolated DNA from another genetically different bacterium. This process was first demonstrated in Streptococcus pneumoniae by F. Griffith in 1928 and O. Avery in 1944. This experiment also showed that DNA was the genetic material.
This experiment of bacterial transformation is also used to map genes in certain bacteria. In this experiment, DNA from one strain, i.e., Donor, is extracted and purified. DNA is then added to a suspension of another bacteria of different strain.
The recipient cell takes up the DNA which undergoes genetic recombination with the parts of the recipient chromosome (DNA) forming a Recombinant chromosome. These recipient cells are now known as Trans-formants.
Although most species of bacteria can undergo transformation, the wild type E. coli is not readily transformable. The efficiency of taking up DNA can be increased by using some special treatment of recipient cells to make them competent.
When a piece of DNA has been taken up by’ the bacteria, it will pair with the homologous segment of DNA of recipient cell. Here the bacterial genome is circular and the inserted foreign DNA is linear, and so the single crossover will make the circular DNA a long, linear chromosome.
This linear chromosome in the bacteria fails to replicate—thus leading to the death of the bacteria. But the double crossover between the circular recipient DNA and the linear donor DNA forms an intact circular recombinant bacterial chromosome and a residual linear piece of DNA which, ultimately, undergoes degradation (Fig. 5.6).
Bacillus subtilis and Streptococcus pneumoniae can be easily transformed into a test tube. Again, the uptake of DNA in the recipient bacterial cells is not species-specific, because bacterial cells may take up DNA from any species—even from mammals.
However, the transformation is possible if the donor DNA shows homology or, in other words, efficient transforming activity is found between the DNA of closely related species. It is interesting to note that the foreign DNA is single-stranded in the bacterial cell which may occur either during entry or inside the cell. Only one strand is conserved and the other is degraded.
Transformation can be used to determine gene linkage, gene order and map distance. The principle of this process of transformation is the following. Say, it has been found in an experiment that the gene x is transformed to x+ with a frequency of 5%.
Now we want to know whether these genes are linked. Then the donor DNA with x+y+ is used to transform cells with x y genotype. If the two genes are widely apart from one another, they will be found on different fragments during extraction of DNA.
Since the probability of x+ trans-formant is 5%, i.e., 0.004, and the probability of other genes (y+) is also 0.005, the chance of occurring double transformation is 0.005 x 0.005 = 0.0025, i.e., 25 in 10,000 cells.
But if the two genes (x+, y+) are close enough so that they are carried on the same DNA fragment, the frequency of simultaneous transformation (co-transformation) or double transformation will be more. Another factor has also been noted which controls the transformation frequency. It the concentration of DNA used in transformation (transforming DNA).
The decrease in the frequency of double transformation is found with the decrease in the concentration of DNA. Linkage between the two genes can be mapped with the help of the bacterial transformation.
In one genetical experiment, recipient’ cells having x–y– genotype have received transforming DNA of x+y+ genotype (Fig. 5.7) and the resulting trans-formant may be:
The second and third group show single trans-formants and the first group shows double trans-formant. As the number of double trans-formant is high, linkage between the genes is present of the total number of trans-formants (500), the number of recombinants are 130 + 90 = 220. So, the linkage distance will be 220/500 = 0.44 or the map distance between the genes x and y is 0.44.
From this linkage distance, gene order can also be determined.
ii. Conjugation:
Lederberg and Totum first observed conjugation in E. coli in 1946. They studied two strains of E. coli which differ in some nutritional requirements. The first strain consists of the genotype methionine–, biotin–, threonine+, leucine+, thiamine+ which does not grow in the minimal medium.
The second strain with the genotype methionine+, biotin+, threonine–, leucine– and thiamine– also does not grow in the minimal medium unless threonine, leucine and thiamine are added in the medium. The strains which require some additives to grow are known as Auxotroph’s. Strains which do not require any supplements in the medium are known as Prototrophs.
In the actual experiment—when the two strains are plated separately in the minimal medium—no growth is found. But when the two strains are mixed and are plated in the medium, they grow like prototrophic bacteria.
Detailed study shows that cell to cell contact is necessary for the growth of the two strains and, during this process, genetic exchange occurs. This type of exchange of genetic material occurs in bacteria through a special type of mating system called Conjugation in which DNA or genetic substance is transferred from one bacterium to another through a tube.
Again, William Hayes observed that only one cell acts as a donor and the other as a recipient. He proposed that this type of conjugation takes place through some fertility factor (F). The donor cell has the fertility factor so it is designated as F+ and the recipient as F– (Fig. 5.8).
This F factor is present only in E. coli, which is, again, present in the plasmid. Later, another strain with high frequency of recombination has been known which has been designated as high frequency recombination strain (Hfr).
Transduction:
It is the process by which the genetic material is transferred between two strains of bacteria by bacteriophages which are a type of virus that infects bacteria. Most bacterial strains have specific phages. For example, E. coli is infected by phages like T2, T4, T5 and etc. All phages contain either DNA or RNA as a genetic material with a protein coat.
When the phage DNA is injected into the bacteria, it follows two types of life cycle—lytic cycle and lysogenic cycle. In the former, phage takes all the control over the bacteria, breaks down the bacterial chromosome, its reproduction etc. and finally kills the bacteria.
In the lysogenic cycle, phage DNA does not replicate by itself, rather, it integrates into the bacterial chromosome and replicates along with the bacterial DNA. This integrated state of phage chromosome is called Pro-phage. This type of bacteria is known as Lysogenic for that phage. Phages are known as Temperate phage.
This process of transduction can be used in transferring a small piece of DNA in other bacterial strains through temperate phage for the mapping of bacterial genes. Phage can also be used as a vector in the techniques of genetic manipulation.
Importance of E. coli in Genetic Engineering Techniques and Applications:
E. coli has played a very important role in the development of biotechnology with the refinement of the Genetic Engineering methods. In spite of the discovery of large number of living organisms as tools in genetic Engineering techniques, E. coli is the most widely used. K12 strain is used as hosts for recombinant plasmids or phages.
In the different processes of Genetic Engineering such as cloning, characterisation and modification of DNA fragments, E. coli systems are used. As regards biotechnological applications, most of the industrial production of proteins, hormones etc. are prepared within E. coli K12 strains.
Yeast:
For biotechnological applications, another convenient and widely used material is the Brewer’s yeast (Saccharomyces cerevisiae). One important advantage in case of Genetical studies in this material is the Tetrad analysis. Here the products of a meiosis—the tetrad— are present within a single structure. Besides yeast, tetrad analysis can also be easily done in Neurospora crassa and Chlamydomonas sp. etc.
Life Cycle of Yeast:
Yeast is a unicellular fungus which is found in the vegetative state—both in the haploid and diploid states. Two mating types—a and a— occur in yeast. These ascospores of two mating type again follow vegetative cycle through budding.
The fusion of two ascospores of ‘a’ and la’ gives rise to a diploid cell or zygote which may also follow vegetative cycle through budding. With the proper environmental conditions, diploid cells undergo meiosis to produce four ascospores of two mating types (Fig. 5.9).
Petite Mutations:
Of the different mutations in the mitochondrial DNA, petite mutation in yeast has been widely studied. Yeast cells with petite mutations show very slow growth with the formation of small colonies on agar plate. These mutants are deficient in aerobic respiration and possess some defects in the enzyme. Petites require glucose—a fermentable product—as a substrate and can use it in presence of oxygen.
It has been found recently that the inheritance of petite mutants is Non-Mendelian. As petite mutants show deficiency in their respiratory capacity, it can be said that these mutants are due to some change in the mitochondrial DNA.
Again, the mitochondrial DNA of these mutants show some differences in the buoyant density from the normal strain of yeast and there is a change in the G+C value. All these changes are due to some deletions of portions of the mitochondrial DNA.
Cell Cycle in Yeasts:
Cell cycle studies have been done in the two species of yeast such as Saccharomyces pombe and S. cerevisiae. These species are chosen due to some advantages like budding type of division, simple and unicellular form, fast growth rate and amenable to easy and quick biochemical and genetical analysis.
Recently the molecular biological studies regarding the cell cycle are possible due to the isolation of conditional lethal cell division cycle mutants (cdc) in S. cerevisiae.
Cell Division Cycle Mutants:
This is a type of mutation leading to a defect in a particular stage-specific function of the cell cycle. Thus cells with cdc mutants (Fig. 5.10) will accumulate at the same stage forming a synchronous and homogeneous culture. This stage of cdc mutants is known as the terminal phenotype. About 70 genes with cdc-mutants have been found in S. cerevisiae. About 30 genes have been mapped over three chromosomes.
The events of the cell cycle occur in sequential order, each step is controlled by the products of genes. The product of first genes controls the function of second genes, and it goes on in this way, showing several events in the cell cycle. The cdc mutants have a conditional block during cell cycle.
In Fig. 5.11, gene product B (gp B) mediates the reaction leading to the conversion of x into y which is essential for cell division. Again, the function of gene product B is dependent on the gene product A (gp A).
So any alteration in any gene blocks the sequential step of the cell cycle, thus affecting cell division at different steps, wee and whi mutants have been found to be very useful in the fundamental studies of the cell division events. Among wee mutants, the function of wee 1 mutants have been noted. It has been found that cdc 2 mutants cannot enter mitosis.
Actually, wee 1 produces one inhibitor which prevents the premature entry of the cell into mitosis. The allele of wee 1 (wee 2) is found to initiate mitosis again, that means it occurs by a change of function causing acceleration into mitosis.
Thus the cell cycle may be described as a periodically repeating linear set of events with important steps of initiation and completion. With the advent of cdc mutants and the cloning of cdc genes, the nature of the gene product can be determined to understand the mechanism of mitotic events.
Animal Cell in Tissue Culture:
Nowadays, the use of animal cell and tissue culture has been taken as a major tool in many different disciplines—from cell and molecular biology to the applied field of biotechnology.
Large number of cell types can be grown in culture such as fibroblasts, skeletal tissue (bone and cartilage), skeletal, cardiac and smooth muscle, epithelial tissues (liver, lung, breast, skin, bladder and kidney), neural cells, endocrine cells, melanocytes and different types of tumour.
For the proliferation of cells, some precursor cell type is used rather than a fully differentiated cell which would not normally proliferate. At low cell densities, fibroblast cultures contain a fairly uniform population of proliferating cells while, at high cell densities (105 cells/cm2), differentiated non-proliferating cells are found.
When the cell density is reduced by scraping or trypsin sing the cells, fibroblasts start to proliferate again. Besides cell density, other factors like serum or Ca++ ions, hormones, cell interactions are also responsible for initiating proliferation. Thus different conditions are required for propagation and differentiation.
In animal systems, tissue culture was defined as the culture of fragments of explanted tissue where the histological integrity was maintained during culture. Cell culture is the method of culture where the tissue is dispersed mechanically or enzymatically from an explant and is grown as a cell suspension.
Thus organ culture retains histological and biochemical differentiation for several days. They cannot be propagated. On the other hand, cell culture will lose its histological differentiation and biochemical properties.
It can be propagated easily in selective media. Again, cultures grown from embryonic tissues will grow better than mature tissues. Embryonic cell lines are widely used in many basic and applied sciences of cell biology, for example, 3T3 lines (Mouse embryo fibroblasts), MRC 5 and human lung fibroblasts.
Generally non-tumorigenic tissues have a limited life span and tissues from the tumour have the ability to grow continuously in the media.
The morphology of a continuous cell line is characterised by smaller cells, less adherent rounded, reduction in serum dependence, increased ability to proliferate in liquid culture, increased cloning efficiency, greater growth rate etc. Some cells are cultured as a monolayer or glass or plastic substrate.
Primary Culture:
When cells are isolated from a tissue and are grown in culture, they are known as Primary culture. Primary cultures can eliminate many cells which are unable to grow (in vitro). Thus any non-dividing or. slow-growing cells can be avoided in the next subculture.
Primary cultures can be made by dispersing the tissue by the treatment of trypsin (0.25% in case of crude or 0.01-0.05% in case of pure) or collagenase and the cells are grown in liquid media containing glass or plastic substrate.
Subculture:
Monolayer culture is transferred to a fresh media after rinsing the monolayer with PBS (Phosphate-buffered saline) or PBS containing ImM EDTA with cold trypsin (.01-.05% pure) for 30 sec. Trypsin was then removed and cells are sub-cultured to a fresh media.
Contamination Problems:
Sometimes the bacterial contamination is eliminated by the use of antibiotics but it is desirable to culture the cells in antibiotic free media. The chance of contamination can be checked by noting the change in pH of the media (usually a fall in pH), turbidity in the medium etc. If there is any contamination, the flask is to be discarded immediately.
Cultures can also be infected by mycoplasma which can affect growth characteristics and cellular biochemistry. Mycoplasma contamination is very difficult to detect and so some test like fluorescent DNA stain technique is to be made frequently.
Media:
There are several media available in the market which is derived from Eagle’s minimal essential medium (MEM), Dulbecco’s enriched modification (DME) and some complex media like Ham’s F12, CMRL 1066, RPMI 1640, McCoy’s 5A and Iscore’s modified Dulbecco’s medium (IMDM).
Some cells require supplements for rapid growth. For example, Chondrocytes and fibroblasts require lipid supplement, epithelial cells require some growth factors etc. Some cells have some preferential response in certain media.
For example:
i. Human and monkey cells prefer 199, 5A, RPMI 1640, CMRL 1969, MC DB 104 and MCDB 202.
ii. Rat and Rabbit cells prefer 5A, F12 and MCDB 104.
iii. Chicken cells prefer 199, DME, F12K and MCDB 202.
Quantification of Cells:
Total cell numbers were measured by counting cells in haemocytometer and the cell mass was measured by determining dry weight or protein. But there are some cells which remain anchored on the glass plate so, in that case, an indirect measurement has to be made for determining the viability of cells.
Cell Viability:
It is known that viable cells cannot take up certain dyes, whereas dead cell can take up these dyes. Trypan blue (0.4%) is commonly used. In this case, only dead cells will be stained. In case of serum dependent cultures erythrosine B (0.4%) is used. Viability can also be measured indirectly noting its metabolic activity, i.e., Glucose utilisation, lactic or Pyruvic acid production, C02 production etc.
Important Points During Culture:
i. Medium is to be warmed at 37°C and pH is to be stabilized before adding the cells.
ii. Avoid using cells from the stationary phase. This may lead to a long lag phase or no growth.
iii. Usually late log phase cells are better.
iv. It is better to initiate culture with cells of high density. The density of cell is generally used between 5 x 104 and 2 x 105 cells/ml.
v. Optimum stirring rate for a culture vessel and cell line is generally between 100 and 500 r.p.m.
vi. Good yield of cells is achieved if there is a good buffering system (Hepes instead of bio-carbonate), continuous gassing, monitoring of pH etc.
The growth of a cell begins with a lag phase, log phase, stationary phase and, finally, to- the decline and death of cells (Fig. 5.12).
Culture Systems:
(i) Batch culture:
This is the type of culture in which cell are cultured into a fixed volume of medium. As the cells are in the media, nutrients are used up and accumulation of metabolites occur. The culture environment is, therefore, gradually changing and so the cell growth ceases.
The constant optimum environment can be maintained by replacing a fixed volume of the culture with fresh medium or by continuous addition of medium to the culture and the withdrawal of an equal volume of used medium (Perfusion technique). This method is suitable for both monolayer and suspension cells.
(ii) Continuous flow culture:
This system gives the real culture conditions with no variation in nutrients, metabolites or cell number. Here the medium enters the culture with the withdrawal of used medium and cells. In this continuous flow culture, all cells are homogeneous and remain as such for long periods of time.
Here cell yields never attain the maximum level as certain limiting growth factor is used to control the growth rate. It is suitable for suspension cultures and monolayer cells growing on micro-carriers (Fig. 5.13).
(iii) Monolayer culture:
The monolayer culture technique has the following advantages:
1. Medium can be changed completely and even the cell sheet can be washed before adding fresh medium.
2. Total removal of undesired compound in the medium is possible.
3. These methods can be used for all cell types.
4. Many cells or cellular products can be studied more efficiently when attached to substrate.
Cell Attachment:
Cell surfaces and glass and plastic surfaces are negatively charged. For cell attachment, some divalent cations (Ca++, Mg++) or glycoproteins are required for cross-linkage. The glycoprotein is present in serum and other physiological fluids. Cells are attached to the substrate by some electrostatic forces. Surfaces are sometimes coated with collagen (Vitrogen 100, Flow laboratories) and poly-amino acids.
Although cells are attached by electrostatic forces, glass and metal having high surface energies are suitable for cell attachment. Arum-borosilicate glass (Pyrex or Borosil) is better because other soda-lime glasses release alkali into the medium—thus changing the pH.
The capacity of attachment of glass after repeated use can be increased by treatment with 1 mM Magnesium acetate and then autoclaved after washing in distilled water.
Plastic-ware made of polystyrene, polycarbonate, perspex, cellulose acetate etc. can be safely used. Stainless steel and titanium can also be used for cell culture as they are chemically inert. But these should be washed with a mixture of 10% Nitric Acid, 3.5% Hydrofluoric Acid and 86.5% water to remove surface impurities.
Immobilised or Encapsulated Cells:
The immobilisation of cells in semi-solid substances has many applications in biotechnology. The substances used for the formation of beads are gelatin, polylysine, alginate and agarose. Alginate can be cross-linked with Ca++ ions.
Generally 10 mM CaCl2 solution is used. Cells are suspended in isotonic saline solution buffered with Tris (ImM) and 4% Sodium alginate. This mixture is then added drop wise to a solution of isotonic saline, 1 mM Tris, 10 mM CaCl2 at pH 7.4 the beads are formed with a diameter ranging from 2-3 mm. The immobilised cells can be freed by dissolving the alginate in 0.1 M EDTA or 35 mM Sodium citrate.
These beads allow diffusion of nutrients from the medium and any hormones, immuno-chemicals, enzymes, antibodies etc. that are synthesised within the cells. Instead of alginate, sometimes 5% Agarose is used. Cold Agarose solution (40°C) is mixed with cells in suspension.
The mixture is then added to paraffin oil which is then cooled in an ice-bath. The spheres of 80-200 μm are washed and are transferred to the culture medium. Some solutions or media are required for animal cell culture.
(i) Hank’s Balanced Salt Solution:
Autoclave for 15-20 min at 15 p.s.i. Before use pH is adjusted to 7.4 with sterile NaOH and sterile 1M Hepes.
Glucose, if necessary, should be autoclaved separately at 100g/l and diluted 1: 100 to give 1-0 g/l.
(ii) Phosphate-Buffered Saline, Solution A (PBSA):
KCL . . . 0.20 g
KH2 PO4 . . . 0.20g
NaCl . . . 8.00g
Na2HPO47H2O . . . 2.16g
Water to 1,000 ml.
Autoclave it before use. Used as a Washing solution.
(iii) Trypsin:
2.5g (crude Difco 1/250) or O.lg (3X recrys- tallised, Sigma) in PBSA or Hank’s solution (1,000 ml).
Growth Characteristics of Animal Cells:
Animal cells may be of two types: primary and transformed. Primary cell cultures are initiated from animal tissue, skin or any embryo genic tissue. But they do not grow for indefinite period in culture. After a few generations—say 100—the growth will stop.
These cultured cells may grow for indefinite period if they undergo some change; i.e., transformed to behave like tumour cells which normally grow indefinitely. Here, transformation of cells means the change in the habit of growth, i.e., cells become immortal. Most of the animal cells during culture do not undergo any differentiation, so these cells remain undifferentiated.
One of the most commonly used undifferentiated cells is the HeLa cell, which grows continuously in culture. HeLa cell was first obtained from a malignant tumour—a carcinoma of the uterine cervix—in 1952. The undifferentiated normal cell is the fibroblasts.
The muscle precursor cells (myoblasts) have been transformed in the laboratory which then grow indefinitely as single cells. Teratoma cells, i.e., cells originated from a tumour, can give rise to many different cell types. But there are some cells like nerve, kidney, etc. which do not undergo any transformation or, in other words, they do not grow indefinitely.
Viruses:
These are small parasites of prokaryotic and eukaryotic cells which cannot reproduce by themselves. A virus consists of an outer protein coat (capsid) within which is packed the genome. The capsid plus the nucleic acid is known as nucleocapsid.
The genome of virus is either DNA or RNA, but not both. DNA or RNA may be either single-stranded or double- stranded. The virus that attacks the bacteria is known as bacteriophage or phage (Fig. 5.14).
Viruses that attack animal or plant cell are known as Animal viruses or Plant viruses. There are some viruses which can grow in insect and plant or insect and animal. The coat protein (capsid) of a virus is either arranged in the form of a polyhedral in shape or as a helix.
In some virus, there is an envelope of lipids and proteins outside the nucleocapsid— example, Influenza virus. This envelope is formed from the membrane of the host cell. The number of virus particles, present in a host cell can be measured by counting the number of lesions in the cell which is known as plaque assay technique (Fig. 5.15).
DNA Viruses:
In these types, the genome is either single or double-stranded DNA, which may be circular or linear. The replication of DNA occurs in different ways. One important mechanism of replication of viral DNA is by rolling circle method. It is found in the E. coli phage φx 174. On entering of the viral DNA into the host, single-stranded closed circular DNA starts synthesizing complementary strand.
The synthesis starts with an RNA primer produced by E. coli primase. DNA polymerase III then starts DNA synthesis in continuous chain. When the circle is complete, the RNA primer is excised and the gap is filled by polymerase I, and then the open point is sealed by ligase.
Now DNA consists of a double-stranded structure, one original strand (plus strand) and the other newly constructed complementary chain (minus strand). Now this strand is known as Replicating form.
Then a nick or break is formed near the origin site of + strand. 5′ end then starts separating from its other strand. In this way the continuous DNA synthesis starts at the 3′ end as the 5′ end starts separating. 5′ end forms a long single-stranded tail which lies in the cytoplasm.
Its 3′ end lies in the duplex ring. When the DNA synthesis is complete over the tail, endonuclease cuts the tail near the origin site (O) eliminating the” tail from the ring. These single-stranded ends of the tail overlap and ligase action produces a closed, daughter, double-stranded ring, called a Daughter replicating form.
The formation of the infectious single-stranded rings occurs in the same way. However, there are several types of mechanisms of replication in DNA viruses.
RNA Viruses:
Here the genome is single-stranded RNA, for example, Tobacco mosaic virus, RNA bacteriophages of E. coli, Polio virus etc. in some single-stranded viruses like influenza, Rous sarcoma, murine leukemia viruses etc., the genome consists of separate pieces of different lengths.
Double-stranded RNA is found in neo-viruses of mammals. But, in all cases, RNA remains in the linear form. The replication of the genome takes place through a replicating enzyme, known as RNA-directed RNA polymerase. This enzyme is hybrid in nature in the sense that one sub-unit is contributed by the parasite and the other sub-unit is produced by the host.
RNA tumour viruses (Retro viruses) contain an enzyme known as RNA-directed DNA polymerase, i.e., Reverse transcriptase. This enzyme helps in the formation of DNA from RNA. Such viruses are responsible for the malignant transformation of cells and, thus, is associated with the origin of a number of different types of cancer in rats, mice and humans.
Viriods:
These are minute self-replicating particles that cause a variety of diseases in plants. Here only naked RNA is present, no protein capsule or coat is present. These viriods are found to be associated with the nucleus of the host.
Plant Cells in Culture:
The refinements of technique of tissue culture have led to the use of this technology for the improvement of new plants. The commercial use of tissue culture has been widely utilised with the help of shoot tip propagation and somatic embryogenesis. Thus this technique can also be used for rapid propagation of elite plants. Tissue culture is also a source of producing genetic variability.
Somaclonal variants and gametoclonal variants may be used to introduce non-specific desirable variation into food crops, ornamentals and medicinal plants. Several new plant varieties have already been developed through somaclonal variation.
Gametoclonal variation through another culture is also used as an alternative to conventional breeding and also helps to make some genetic manipulation in the commercially viable plants. Development and use of haploid plants have already been extensively done in China.
The isolation and selection of mutants and protoplast fusion establish a method of incorporating foreign genes or organelles to attain desirable somatic hybrid. Cell culture techniques including the use of some immobilised systems have accelerated the production of secondary metabolites for the food and pharmaceutical industry.
The application of all these technologies have opened a new vista in plant and agricultural sciences.