In this article we will discuss about: 1. Methods of Cloning, 2. Cloning of a Specific Gene, 3. Cloning of a Specific Gene, 4. Expression of Cloned Genes, 5. Cloning of Genes in Eucaryotic Cells and 6. Precautions to be taken during Experiments of Genetic Recombination.
Methods of Cloning:
In the past few years, an increasing number of laboratories have undertaken experiments of genetic recombination in vitro. These experiments consist in incorporating heterologous DNA in a stable manner in a bacterial cell (generally the strain of E.coli K12), through a vector which can be, either a plasmid, or a phage such as phage , i.e. genetic material capable of existing independently of the bacterial chromosome and of replicating autonomously.
For example, if a plasmid is used (i.e. a circular double-stranded covalently closed DNA molecule present in the bacterial cell independently of the chromosome), the principle of the experiments will consist in isolating the plasmid from the bacterial cell, splitting this DNA molecule by means of a very specific enzyme (a restriction endonuclease), joining the cleaved plasmid DNA to the heterologous DNA to be cloned and finally re-introducing — thanks to the transformation process — in the bacterial cell, the plasmid carrying the heterologous DNA.
Among the most frequently used plasmids, one can mention those made in vitro using the DNA recombination techniques, and which often belong to the Col series, because they contain genes coding for the production of a colicine (an extracellular protein, having antibiotic properties, produced by E.coli).
The plasmids used for the cloning of genes generally have the following properties: their size is comparatively small; they possess at least one single site sensitive to a restriction endoculease; they contain one (or several) gene(s) which allow the selection of the cells transformed by the plasmid; they are present at the concentration of about 20 molecules per cell, but in presence of chloromphenicol their replication leads to the accumulation of 1 000 to 2 000 circular plasmid DNA molecules per cell; they can be easily isolated from bacterial cells and can also be easily reintroduced in them.
The diagram of fig. 6-51 shows the main steps allowing the cloning of heterologous genes in a bacterial cell. Owing to its large size, the bacterial DNA is very sensitive to shearing forces, with the result that it is cut into fragments during the extraction of DNAs, while the plasmid DNA much smaller in size, present in the form of circular (covalently closed) double- stranded super-coiled molecules, remains intact and can be easily separated from the bacterial DNA by centrifugation in a density gradient, in presence of a molecule which intercalates between the bases of the DNA, ethidium bromide.
The physical constraints existing in a covalently closed circular DNA, molecule are such that the quantity of ethidium bromide bound will be limited. These constraints do not exist in a linear molecule and the quantity or ethidium bromide bound per unit DNA length will be much greater.
Compared to the density of the circular complexes, the density of the linear complexes will therefore be sufficiently lowered to permit their separation during an isopicnic sedimentation (the decrease in density of the DNA is proportional to the quantity of ethidium bromide bound per unit length).
To cleave the plasmid DNA and the heterologous DNA one can use a restriction endonuclease producing cohesive ends which on the one hand, will subsequently facilitate the joining of the heterologous DNA fragment with the plasmid by means of polynucleotide ligase, and on the other hand will permit — with the help of the same restriction enzyme — the excision of the inserted DNA from the plasmid (if, for example, one wants to study the structure of the DNA fragment inserted).
But one can also use other restriction enzymes (which do not produce cohesive ends, e.g. Hae III), and achieve cyclization after addition of poly dA at the 3′ ends of the heterologous DNA fragments and of poly dT at the 3′ end of the cleaved plasmid DNA, which permits pairing of poly dA and poly dT segments, and then covalent joining of the fragment and the plasmid with the help of polynucleotide ligase; this method offers the advantage of avoiding the cyclization of the plasmid without insertion of foreign DNA, with the result that practically all the transformed bacterial cells, selected using genetic marker of the plasmid, will contain a hybrid plasmid (i.e. having inserted heterologous DNA).
In general, in optimal conditions, one E.coli cell out of 105 or 106 will be transformed by the plasmid. It must be noted that there is no minimal size for the heterologous DNA inserted in a plasmid, and that this size can reach at least 40 x 106 daltons, i.e. about 60 000 base pairs (= 60 kb).
Moreover, the insertion of foreign DNA in plasmids of the Col series does not affect the number of plasmid molecules which can be present in the cell, so that the heterologous DNA inserted will be present also at the concentration of several copies in the transformed bacterial cell. Lastly, under the effect of chloramphenicol one can have an amplification such that the circular DNA of the hybrid plasmid represents 40 to 50% of the totality of the DNA of the cell.
The DNA of the bacteriophage (47 000 base pairs = 47 kb) is often used as cloning vector because it offers some advantages over the bacterial plasmids. About 15 kb of the central part of the genome of this phage are not essential for its survival and can be replaced by heterologous DNA to be cloned.
One can carry out in vitro the encapsidation of the recombinant DNA obtained and thus considerably increase the infection efficiency of this DNA. The encapsidation mechanism of the phage is such that only DNA molecules having a final size of about 47 kb will be encapsidated, which brings about a spontaneous selection of DNA molecules, having inserted a heterologous fragment of about 15 kb.
Cosmids are small plasmids which contain an encapsidation signal (the “cos” site of phage ), an origin of replication and a gene of resistance to an antibiotic.
They therefore combine the advantages of the bacteriophage (efficiency of infection of the encapsidated form) and those of bacterial plasmids (autonomous replication and ease of selection). Furthermore, one can insert in cosmids, fragments of heterologous DNA of larger size (up to 40 kb).
These two vectors of cloning (phage and cosmids) are often used for constructing genomic “banks” or “libraries” which are collections of recombinant phages or plasmids that together comprise the totality of the sequences of a given genome. It is clear that the greater the size of genomic DNA fragments inserted, the smaller the number of clones needed to cover the totality of the genome: this is the special advantage of cosmids.
Cloning of a Specific Gene:
The problem is to select from a vast population of transformed cells, a cell which contains a hybrid plasmid carrying a particular heterologous gene. This is a serious problem, especially if the starting heterologous DNA is complex and if the fragments generated by the restriction enzyme are numerous. It is clear that either there must be a phenotypical property associated with the gene cloned, or one must have an appropriate probe.
If one clones a gene coding for an enzyme for example, one can transform a bacterial mutant lacking this enzyme and thus select the appropriate transformed cells on the basis of the restoration of the corresponding enzymatic activity.
It was thus possible to clone the genes of the tryptophan operon, or the gene of the ligase of E.coli, starting from DNA fragments resulting from the hydrolysis of the whole chromosome of E.coli. But if one wants to clone a eucaryotic gene, it is often preferable to clone first the DNA copy (cDNA) of the mRNA corresponding to the gene sought.
The size of these cDNAs is indeed generally much smaller than that of their genomic counterpart due to the absence of introns. One therefore constructs a cDNA “library” by inserting, in a plasmid or in a vector derived from bacteriophage (e.g., gt 10), cDNA copies of the entire population of cytoplasmic mRNAs extracted from the cells expressing the gene concerned.
These copies are made with the help of the reverse transcriptase which synthesizes the cDNA strands complementary to the mRNAs starting from oligo dT primers previously hybridized to the poly A tails of mRNAs.
After alkaline hydrolysis of the RNAs, double-stranded cDNA molecules are then obtained by action of DNA polymerase I of E.coli which makes strands complementary to those synthesized during the first step. The synthesis is “self-primed” at the 3′ end of the single-stranded cDNA which folds back upon itself (see fig. 6-31a).
A digestion by nuclease S1 (specific of single stranded nucleic acids) finally eliminates the loops at this end and gives perfectly double-stranded cDNAs. A cDNA library is said to be complete if it contains at least one bacterial clone for each starting mRNA.
There are several methods to identify the clones sought. If the cDNAs have been inserted downstream a bacterial promoter (as in the case of gt 11), one can analyse (or “screen”) the “library” by the expression of the cDNAs. This is done by immunological identification of the producer clones; antibodies specific of the corresponding protein will react selectively with the relevant clones which will thus be labeled.
The success of this “screening” technique however depends on several factors: the cDNA must be inserted in the correct orientation with respect to the bacterial promoter of the vector, in order to permit the transcription of its coding strand; the translation of the messenger thus produced must take place in the correct reading frame; lastly, the tertiary structure adopted by the synthesized polypeptide must reconstitute the antigenic determinants (epitopes) recognized by the antibodies.
A second approach, independent of these prerequisites, is based on recent developments in the micro-sequencing techniques of proteins and in the production of synthetic oligodeoxynucleotides. With the knowledge of the partial amino acid sequence of the protein one can, on the basis of the genetic code, deduce (taking into consideration the fact that the code is degenerate) the corresponding nucleotide sequence and carry out its chemical synthesis.
These oligonucleotides, labeled by 32P, are then used as radioactive probes to identify, by hybridization with the complementary sequences, the bacterial clones containing the cDNA sought.
The cDNAs thus isolated can in their turn serve as probes to look for the corresponding genes in a genomic “bank”.
Expression of Cloned Genes:
These hybrid plasmids represent not only an abundant source of inserted heterologous DNA, but also allow the production – by the bacterial cell – of large quantities of products specified by the genes inserted, particularly when the cloned genes are from the same bacterium or another procaryote: for example, the cloning of the tryptophan operon of E.coli in a plasmid of the Col series has allowed the induced synthesis of such large quantities of the five enzymes of this operon that they represented 40% of the total proteins of the cell.
But when genes of eucaryotes are cloned in E.coli plasmids, it is observed that the synthesis — by the bacterium — of the products corresponding to these genes is very small, or even nil, most probably because of differences between the systems controlling transcription and translation in procaryotes and eucaryotes (for example in the initiation and termination of transcription and translation) or because of the absence of intron splicing systems in procaryotes.
Some of these problems can be solved by the insertion of a cDNA (DNA complementary to the mature mRNA) directly downstream a functional procaryotic promoter in the plasmid. In this manner one has obtained in bacteria, the synthesis of proteins of different origins: animal (growth hormone, somatostatin, insulin, interferon), plant (large sub-unit of ribulose 1, 5 bisphosphate carboxylase) or viral (antigen of the hepatitis B virus, or of the foot and mouth disease virus).
It must be emphasized however that the biological activity of some of these proteins can depend on subsequent modifications (specific proteolytic cleavages in the case of precursors or glycosylation) which the procaryotic host cannot perform.
Cloning of Genes in Eucaryotic Cells:
At the beginning, the cloning of genes was mostly carried out using E.coli, but the principles on which this process is based are also applicable to animal and plant cells. Viruses or plasmids capable of introducing genes in the chromosomes of eucaryotic cells were needed; it was therefore envisaged to use viruses derived from Simian virus 40 (SV 40).
As far as the plant kingdom is concerned, it was shown that a fragment of the DNA (T-DNA) of a plasmid (Ti plasmid) of the bacterium Agrobacterium tumefaciens (responsible for the formation of a tumour called Crown-Gall) could be transferred to plant cell chromosomes.
Moreover, one could insert, in this T-DNA, genes of bacterial origin (e.g., gene of resistance to an antibiotic) or plant origin (e.g., gene of the small sub-unit of ribulose 1,5 bisphosphate carboxylase) and thus obtain their transfer to a tobacco protoplast; since in various species (including tobacco) one can regenerate an entire plant from a protoplast, one could observe, starting from transformed protoplasts, the presence of the gene inserted in diverse tissues of the transformed plant, observe its expression and note that this expression is controlled by factors (such as light) which exert their regulatory effects at the transcriptional level in plants.
Some recent techniques enable the direct introduction of DNA into plant or animal cells without the help of any plasmid or viral vector (direct transfer of genes).
Advantages of the Cloning of Genes:
Cloning of genes provided for the first time, large quantities of specific DNA fragments, in other words genes, especially of higher organisms. These DNA fragments can be used as probe and permit, by hybridization, the detection and dosage of the corresponding mRNAs in cells which are at different stages of their development or placed in diverse physiological conditions.
On the other hand one can, thanks to the cloning of genes, undertake the study of the structure of genes and chromosomes of eucaryotes, and the study of the control mechanisms of gene expression in higher organisms.
But, beside the great advances that may be expected in the domain of fundamental knowledge in molecular biology, the cloning of genes can lead to extremely important applications.
In the field of agronomy, one can envisage transferring to plants:
1. A gene responsible for resistance to a herbicide (isolated for example from a microorganism) thus allowing the protection of the cultivated plant when weeds are eliminated by the herbicide;
2. A gene responsible for resistance to a pathogen (e.g., fungus, virus, insect). It has been possible to obtain plants protected from infection by a virus (e.g., Tobacco Mosaic Virus) by introducing into these plants the gene coding for the coat protein of this virus, or plants protected from attack by insects by introducing into these plants the gene of a bacterial toxin having insecticide properties;
3. Genes improving the growth of plants, for example, enabling a more efficient photosynthesis, or permitting atmospheric nitrogen fixation (although, in the microorganisms studied, this process implies the coordinated expression of 17 genes);
4. Genes improving the nutritional value of plants, for example, providing seed storage proteins richer in some essential amino acids, like lysine.
This assumes that the gene is not only introduced in the plant, but also expressed in the right tissue (e.g., the seed) and at the right time.
In medicine, the production of therapeutically important substances, by (bacterial, animal, human) cells in culture after introduction of the corresponding gene, is a major application of genetic engineering. Among substances which are already being produced or under study, one may cite: insulin (used in the treatment of diabetes), growth hormone (used against some forms of dwarfism), interferons (anti-viral action), the plasminogen activator (active against thromboses, because plasmin is an enzyme which degrades the fibrin of clots), α-anti-trypsin (active against emphysema, because it inhibits elastase which digests the elastin of the pulmonary conjunctive tissue), the blood factors VIII and IX (against hemophilia A and B, respectively), various viral antigens, like those of hepatitis B or foot and mouth disease (for the preparation of vaccines), etc.
It must be noted that the advantage lies not only in the production of large quantities of human proteins, which will not cause allergies (unlike for example, pig insulin used till now), but also in the fact that the products are free from contaminants, especially of viral origin, present particularly in substances prepared from human blood, particularly when it becomes necessary to pool the blood of many donors to prepare appreciable quantities of these products (for example in the case of antihemophilic factors), which increases risks (several hemophilic patients were thus contaminated by the hepatitis or Aids virus).
Genetic engineering techniques have also led to major advances in the diagnosis of hereditary diseases. After action of a restriction enzyme on the DNA of a patient, one visualizes the DNA fragments produced at the genetic locus of the disease by hybridization with an appropriate radioactive probe (DNA segment or synthetic oligonucleotide corresponding to this locus or to a portion of it).
The comparison of the size of DNA fragments thus revealed, with those produced in the same manner from a healthy subject permits the detection of anomalies if any: deletions or even, if the hybridization conditions are sufficiently selective (or “stringent”), point mutations.
When the gene responsible for the disease in question is not known, one may utilize the existence in the genome, of zones whose sequence may vary from one individual to another (polymorphic zones or mutation hot points). These zones, characterized by the appearance or disappearance of cleavage sites for a given restriction enzyme, therefore present a restriction fragment length polymorphism (RFLP).
Thus, if one has a probe which can reveal such a polymorphism near the presumed locus of the disease, one can associate the appearance of the disease in an individual with a restriction profile characteristic of the DNA of this individual. It is then possible, by analysis of the RFLP of the DNA of parents and descendants of this individual, to follow the segregation of the gene responsible for the disease, with the help of this probe.
It has also been envisaged to introduce the intact gene, into animal or human cells deprived of a particular enzymatic activity because of a mutation in the corresponding gene, and thus restore the activity lacking in these cells.
Important applications of genetic engineering are also possible:
i. In the field of environment, for example for the biodegradation of toxic products, or for microbiological extraction of various metals from low grade ore.
ii. In the field of energy, for example, for the production of methane and methanol, or for the recovery of crude oil not easily accessible by conventional extraction methods.
Precautions to be taken during Experiments of Genetic Recombination:
It has been said that living species could preserve their identity for a multitude of generations thanks to the presence of natural barriers, and that cloning of genes, by violating these barriers, could create new forms of life, dangerous to man and his environment.
Experiments on genetic recombination in vitro and cloning of genes, as well as the possible risks which could endanger humanity, have been the object of many controversies. Scientists carrying out such experiments in the United States as also in Europe have decided to lay down a number of precautionary measures.
While we will not discuss them here, we will just mention that such measures concern on the one hand, the laboratories where the experiments are carried out and on the other hand the plasmids and bacteria used. As regards the laboratories, one must naturally work in conditions which exclude risks of dissemination as in the case of experiments with pathogenic bacteria and viruses.
As far as the plasmids are concerned, one must preferably use plasmids lacking genes which permit bacterial conjugation; this will reduce the risk of dissemination of hybrid plasmid molecules in our environment.
Regarding bacteria, strains of E.coli K12 were developed, having a number of mutations and necessitating in particular, diaminopimelic acid (a constituent of the bacterial cell wall). If this compound which is indispensable to the mutant is not provided in the culture medium, the cells die and their propagation outside the laboratory is ruled out.