In this article we will discuss about the Multiplication of Host Cell and Cloning of Genes.
Introduction to Multiplication of Host Cell and Cloning of Genes:
The host cell containing foreign DNA in addition to its own DNA is allowed to multiply forming a clone having millions of identical cells. Each member of the clone contains in addition to its normal DNA segment of foreign DNA joined to a DNA of cloning vehicle.
By this process a piece of genet information can be transferred or introduced into DNA genome of recipient species where it could never occur naturally, hence a new organism can be created.
In general, simply cloning a piece of DNA is not enough. The information in DNA must be converted into a useful product. To make a product useful the information in DNA is usually transferred from the gene to the site where a new protein molecule is synthesized through gene expression. A number of problems may be associated with the expression of cloned genes in the recipient cell.
Recombinant DNA works when the host cell expresses protein from recombinant gene. A significant amount of recombinant protein will not be produced by the host unless expression factor are available in the cell. Protein expression depends upon the gene surrounded by a collection of signals which provide instructions for the transcription and translation of the gene by the cell.
These signals include the initiators, promoters, the ribosome binding site and the terminator. All bacteria genes contain such signals but the signals associated with eukaryotic genes are not recognised in bacteria. One solution to this problem is to insert the eukaryotic DNA into the middle of bacteria gene, thus using the existing regulatory signals.
But the product of translation will then be a hybric protein with additional aminoacid sequences (derived from bacterial gene) at either end. Expression vectors, in which the foreign DNA is inserted, contain these signals. The signals are species specific. In the case of E. coli these signals must be E. coli signal as E. coli is unlikely to understand the signals of human promoters and terminators.
Problems are encountered if the gene contains introns or it contains signals which act as terminators in bacterial host. This results in premature termination and the recombinant protein may not be synthesized properly and folded correctly, or it may even be degraded. Production of recombinant proteins in eukaryotic systems generally takes place in yeast and filamentous fungi.
The use of animal cells is difficult due to the fact that many needs a solid support surface, unlike bacteria and have complex growth needs. However, some recombinant proteins are too complex to be produced in bacteria and so eukaryotic cells must be used.
Gene Bank or Genome Bank:
Gene bank is also called genomic library. It refers to the complete collection of cloned DNA fragments which comprises the entire genome of an organism. Gene bank is developed by a short gene experiments where genome of a cell is cloned in the form of random and unidentified clones.
The cloned DNA are produced by the following method:
1. Isolation of DNA fragments to be cloned.
2. Isoining the fragments to a suitable vector (usually phage).
3. Introduction of recombinant DNA into the host cell at high efficiency to get a large number of independent clones.
4. Selection of the desired clone.
5. Use of clones for the construction of gene bank.
For fractionation of DNA (about 10 kb or more in length) to be cloned is important in approaching the maximum cloning capacity of the vector used. DNA fragments of size not suitable for cloning, if ligated to vector, will lower the efficiency of introduction of recombinant DNA into cells. DNA fragmensts of suitable size are obtained through digestion with several restriction enzymes and by gel electrophoresis.
These fragments are cloned without any attempt to select particular sequences. Bacteria lining recombinant DNA are plated on culture media to give rise to individual colonies. If the sizes of the fragments is small, more colonies are needed to be grown.
cDNA Clone Bank or cDNA Library:
A complimentary copy of DNA developed on mRNA molecule is known as copy DNA or cDNA. The cDNA molecule inserted into a suitable vector, transforms a bacterial cell in such a way that it does not disrupt the normal functions of the host cell.
The transformed bacterial cell containing a plasmid with cDNA is known as cDNA clone. It is difficult to get cDNA from double stranded DNA molecule. Therefore, cDNA clones have been prepared from mRNA sequences of eukaryotic cells. The procedure for obtaining cDNA to develop a library is illustrated in Fig. 24.13.
An eukaryotic cell may contain thirty thousand different mRNA sequences. The cDNA clone bank is defined as a population of bacterial transformants, each containing a plasmid with single cDNA insert and with sufficiently large number of individual transformants so that every mRNA molecule is represented at least once in the bacterial population.
A complete bank is one that contains about 5,000 to 10,000 clones of the sequences. A desired clone is screened, identified, isolated and used as and when required.
The benefit of cDNA clone bank are as follows:
1. Preparation of cDNA clone is easy in RNA viruses for example, influenza virus, reovirus etc.
2 Screening of cDNA is easy as it contains less number of cDNA prepared from mRNA of the eukaryotic cells that may contain between 10,000 to 30,000 mRNA molecules hence there is a possibility of getting 10,000-30,000 DNA fragments.
3. As the genes in prokaryotes do not contain introns (the intervening non-transcribed regions of DNA sequence which interrupt the transcript of most of the genes), the DNA clones possess an uninterrupted copy of mRNA molecules.
It may be attributed to the facts that the prokaryotes produce splicing enzymes for ligation process just to remove the sequences of interons. In eukaryotes, however, the interons split the transcribed DNA into many fragments which are known as exons.
4. Following hybridization process, the nucleotide sequence of the gene interrupted by introns may be determined from a genomic clone by comparing it with mRNA transcripts. Therefore, a cloned cDNA is a most suitable probe for hybridization.