Introduction to Molecular Biology: From Cells to Atoms - Anthony Rees, Michael Sternberg 2002

Nucleic Acids and Genes
Genetic Engineering: Gene Cloning

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Fig. 30.1.

Gene cloning is a Procedure involving the isolation and Amplification of individual genes in recipient Cells, whether prokaryotic or eukaryotic. These cells containing the gene of interest can be used to obtain either a) a large quantity of the protein encoded by that gene, or b) a large quantity of the gene itself in a highly purified form. Currently, two main strategies are used for gene cloning. In the first approach, genomic DNA is first randomly cleaved using Restriction Endonucleases (see below) into small fragments roughly the same length as the gene. Each fragment is then introduced via a vector into a recipient Cell, which subsequently multiplies, thereby amplifying the gene or genes contained within that fragment. We will not return to this method again. In the second approach, DNA copies (cDNAs) are synthesized from Messenger RNA isolated from uniform cells of an Organism; these cDNAs are then introduced into recipient cells—on average, one gene per cell—and amplified in the same manner as in the first case.

Recipient cells, i.e., the cells chosen for gene cloning, can be either prokaryotic or eukaryotic. Bacteria are most commonly used for this purpose because they are easily obtained in large quantities. If, however, a gene has been isolated from mammalian cells using the first method, it must be cloned in Eukaryotic cells, such as Yeast, due to the inability of DNA containing introns (Chap. 27) to be expressed in a Prokaryotic Cell. Let us now examine the second method. Messenger RNA (mRNA) is extracted from cells or Tissues that express the gene of interest. For example, to clone the proinsulin gene, pancreatic $eta$-cells should be used, as they are characterized by a high concentration of proinsulin mRNA. Total cellular mRNA can be fractionated using sucrose gradient centrifugation, although these fractions overlap considerably. This rough purification reduces the number of contaminating mRNA molecules in the preparation. DNA copies are then synthesized from the mRNA molecules falling within the desired molecular weight range (for a protein the size of proinsulin, the corresponding mRNA molecule is approximately 25,000 NUCLEOTIDES, or 25 kb, in length).

The synthesis of DNA copies is catalyzed by an enzyme called Reverse Transcriptase (also known as RNA-dependent DNA polymerase), which is usually isolated from appropriate RNA-containing Viruses. For this enzyme to initiate synthesis, a short (about 10 nucleotides) single-stranded DNA primer is required; oligo(dT) is typically used for this purpose. The primer DNA spontaneously forms a double-stranded complex with the poly(dA) stretch that is always present at the 3'-end of eukaryotic mRNA molecules (Chap. 22). Upon completion of the copying stage, the original RNA template is degraded (depolymerized). This depolymerization is achieved via alkaline Hydrolysis. DNA strands are resistant to alkali Treatment, whereas RNA is completely depolymerized. The resulting DNA is single-stranded (ss), with only a small hairpin loop formed at one end of the molecule. Such a hairpin forms because the 5'-end of most mRNAs contains a sequence where one half is complementary to the other (a palindrome), which is also copied into the cDNA. Thus, the terminal region of the cDNA strand containing this sequence can fold back on itself to form a loop and a short double-helical segment.

Double-stranded (ds) cDNA is produced by extending the ss-cDNA into a fully double-stranded form using the enzyme DNA polymerase I. Although this enzyme also fundamentally requires a primer to function, the short double-helical segment formed by the hairpin readily serves this purpose here. One end of such ds-cDNA still retains a single-stranded loop, which is removed using the enzyme S1 nuclease. This enzyme cleaves the loop and also trims the DNA strands by removing any remaining ss-DNA. Following this treatment, the cDNA is ready for insertion into a vector.

A vector is essentially a molecular "taxi" capable of carrying foreign DNA into a bacterial cell in such a way that it can replicate there. There are two primary types of vectors: bacterial Plasmids and Bacteriophages. Here we will discuss only the first type.

Plasmids are naturally occurring extrachromosomal elements consisting of closed, circular ds-DNA molecules (Chap. 26). They are capable of replicating independently of the bacterial genomic DNA. Plasmids often carry genes whose protein products confer resistance to specific Antibiotics. This property is used to separate bacteria containing plasmids ("+" bacteria) from "-" bacteria lacking plasmids. To incorporate cDNA into a plasmid, the closed circular plasmid must be "opened." To achieve this, plasmids are treated with restriction Enzymes.

A restriction enzyme (Chap. 18) cleaves ds-DNA at specific nucleotide sequences known as restriction sites (usually short palindromic sequences); different restriction enzymes recognize different palindromes. Naturally occurring plasmids often contain many such sites for each restriction enzyme. Since, ideally, we require a single Cleavage point, it makes sense to deliberately select or "engineer" plasmids with this characteristic. For example, the plasmid pBR322, widely used as a vector, has only a single restriction site for many restriction enzymes; it also carries genes conferring resistance to ampicillin and penicillin, while lacking certain "non-essential" genes present in its prototype. Only those genes necessary for Bacterial Transformation and plasmid Replication are retained.

Ligation is the process by which foreign DNA is inserted between (or joined to) the two ends of plasmid DNA using an enzyme called DNA ligase. For this operation to succeed, the ends of both the cDNA and the plasmid DNA must be "sticky." This requires single-stranded sequences at the ends that can form a complex with each other via base pairing, thereby holding the two pairs of ends together. This forms what is known as a recombinant plasmid. The procedure described below to accomplish this is called the homopolymer tailing method. There is another procedure known as restriction site ligation, but we will not consider it here.

The homopolymer tailing method is based on attaching short stretches of single-stranded DNA with a regular sequence (homopolymers) to the 3'-ends of the strands forming the ds-DNA. If each such homopolymer consists of a single type of nucleotide, and if two such homopolymers with mutually complementary bases are attached respectively to the plasmid and the cDNA, then the two molecules, when brought together, will anneal to form a recombinant plasmid.

Transformation occurs after the recombinant plasmid is added to the recipient bacterium: the plasmid penetrates the interior of the bacterium and becomes integrated into its life cycle. Since not all bacterial cells in the reaction mixture will be transformed, it is desirable to perform a Selection so that only recombinant bacteria can multiply. Selection can be based on the fact that plasmids confer resistance to certain antibiotics. This means that only those bacteria carrying the plasmids will grow in the presence of the corresponding antibiotic.

Screening is a procedure necessitated by the fact that the initial cDNA preparation contains many different mRNAs, and only a fraction of the plasmids carry the gene of interest. The techniques used for this are highly specialized and will not be discussed here; detailed descriptions can be found in the References provided. Once the desired bacterium has been successfully isolated, it is straightforward to clone and propagate it in culture. This forms The basis of the next stage, known as amplification.

Amplification relies on the fact that a single bacterial cell can synthesize many copies of the plasmid of interest, as well as on obtaining a large mass of cells containing such plasmids. Following Isolation and Purification, the plasmids are treated with the appropriate restriction enzyme, which excises the incorporated copies of the target gene. The gene amplified in this manner can be used for further genetic engineering experiments similar to those listed in the Recommended reading. In addition, bacteria can be utilized to produce the protein product of the gene inserted into the plasmid, provided two conditions are met: a) METABOLISM/31.html">Transcription of the gene occurs, and b) the desired protein is secreted by the bacterium.