2.8A). In contrast, host cells that carry a plasmid-cloned DNA construct produce white colonies on the same medium. The reason for this is that target DNA inserted into a restriction endonuclease site within the multiple-cloning site usually disrupts the correct sequence of DNA codons (reading frame) of the lacZ′ gene and prevents the production of a functional LacZα fragment, so no active hybrid β-galactosidase is produced (Fig. 2.8B). In the absence of β-galactosidase activity, the X-Gal in the medium is not converted to the blue compound, so these colonies remain white (Fig. 2.8A). The white (positive) colonies subsequently must be confirmed to carry a specific target DNA sequence.
Figure 2.8 (A) Strategy for selecting host cells that have been transformed with pUC19 carrying cloned target DNA. E. coli host cells transformed with the products of pUC19–target DNA ligation are selected on medium containing ampicillin, X-Gal, and IPTG. Nontransformed cells and cells transformed with circularized target DNA do not have a gene conferring resistance to ampicillin and therefore do not grow on medium containing ampicillin (recircularized target DNA also does not carry an origin of replication, and therefore, the plasmids are not propagated in host cells even in the absence of ampicillin). However, E. coli cells transformed with recircularized pUC19 or pUC19 carrying cloned target DNA are resistant to ampicillin and therefore form colonies on the selection medium. The two types of ampicillin-resistant transformants are differentiated by the production of functional β-galactosidase. (B) IPTG in the medium induces expression of the lacZ′ gene by binding to the LacI repressor and preventing LacI from binding to the lacO operator sequence. This results in production of the β-galactosidase LacZα fragment in cells transformed with recircularized pUC19. The LacZα fragment combines with the LacZω fragment of β-galactosidase encoded in the host E. coli chromosome to form a functional hybrid β-galactosidase. β-Galactosidase cleaves X-Gal, producing a blue product, and the colonies on the plate appear blue. Insertion of target DNA into the multiple-cloning site of pUC19 alters the reading frame of the lacZ′ gene, thereby preventing production of a functional β-galactosidase in cells transformed with this construct. Colonies that appear white on medium containing X-Gal and IPTG carry the cloned target gene (A).
A number of selection systems have been devised to identify cells carrying vectors that have been successfully inserted with target DNA. In addition to ampicillin, other antibiotics such as tetracycline, kanamycin, and streptomycin are used as selective agents for various cloning vectors. Some vectors carry a gene that encodes a toxin that kills the cell (Table 2.2). The toxin gene is under the control of a regulatable promoter, such as the promoter for the lacZ′ gene that is activated only when the inducer IPTG is supplied in the culture medium. Insertion of a target DNA fragment into the multiple-cloning site prevents the production of a functional toxin protein in the presence of the inducer. Only cells that carry a vector with the target DNA survive under these conditions.
Table 2.2 Some toxin genes used to select for successful insertion of target DNA into a vector
In addition to E. coli, other bacteria, such as Bacillus subtilis, often are the final host cells. For many applications, cloning vectors that function in E. coli may be provided with a second origin of replication that enables the plasmid to replicate in the alternative host cell. With these shuttle cloning vectors, the initial cloning steps are generally conducted using E. coli before the final construct is introduced into a different host cell. In addition, a number of plasmid vectors have been constructed with a single broad-host-range origin of DNA replication instead of a narrow-host-range origin of replication. These vectors can be used with a variety of microorganisms.
Broad-host-range vectors can be transferred among different bacterial hosts by exploiting a natural system for transmitting plasmids known as conjugation. There are two basic genetic requirements for transfer of a plasmid by conjugation: (i) a specific origin-of-transfer (oriT) sequence on the plasmid that is recognized by proteins that initiate plasmid transfer and (ii) several genes encoding the proteins that mediate plasmid transfer. The genes encoding the transfer proteins may be present on the transferred plasmid (Fig. 2.9A), in the genome of the plasmid donor cell, or supplied on a helper plasmid (Fig. 2.9B). Some of these proteins form a pilus that extends from the donor cell and, following contact with a recipient cell, retracts to bring the two cells into close contact. A specific endonuclease cleaves one of the two strands of the plasmid DNA at the oriT, and as the DNA is unwound, the displaced single-stranded DNA is transferred into the recipient cell through a conjugation pore made up of proteins encoded by the transfer genes. A complementary strand is synthesized in both the donor and recipient cells, resulting in a copy of the plasmid in both cells.
Figure 2.9 Plasmid transfer by biparental (A) or triparental (B) conjugation. A plasmid carrying a cloned gene, an origin of transfer (oriT), and transfer genes is transferred by biparental mating from a donor cell to a recipient cell (A). Proteins encoded by the transfer genes mediate contact between donor and recipient cells, initiate plasmid transfer by nicking one of the DNA strands at the oriT, and form a pore through which the nicked strand is transferred from the donor cell to recipient cell. In a cloning experiment, the donor is often a strain of E. coli that does not grow on minimal medium, allowing selection of recipient cells that grow on minimal medium. Acquisition of the plasmid by recipient cells is determined by resistance to an antibiotic, such as ampicillin in this example. If the plasmid carrying the cloned gene does not possess genes for plasmid transfer, these can be supplied by a helper cell (B). In triparental mating, the helper plasmid is first transferred to the donor cell, where the proteins that mediate transfer of the plasmid carrying the cloned gene are expressed. Although the plasmid carrying the cloned gene does not possess transfer genes, it must have an oriT in order to be transferred. Neither the helper nor donor cells grow on minimal medium, and therefore, the recipient cells can be selected on minimal medium containing an antibiotic such as ampicillin. To ensure that the helper plasmid was not transferred to the recipient cell, sensitivity to kanamycin is determined.
Cloning Eukaryotic Genes
Bacteria lack the molecular machinery to excise the introns from RNA that is transcribed from eukaryotic genes. Therefore, before a eukaryotic sequence is cloned for the purpose of producing the encoded protein in a bacterial host, the intron sequences must be removed. Functional eukaryotic mRNA does not contain introns because they have been removed by the eukaryotic cell’s splicing machinery. Purified mRNA molecules are used as a starting point for cloning eukaryotic genes but must be converted to double-stranded DNA before they are inserted into a vector that provides bacterial sequences for transcription and translation.
Purified mRNA can be obtained from eukaryotic cells by exploiting the tract of up to 200 adenosine monophosphates (polyadenylic acid [poly(A)] tail) that are added to the 3′ ends of mRNA before they are exported from the nucleus (Fig. 2.10). The poly(A) tail provides the means for separating the mRNA fraction of a tissue from the more abundant ribosomal RNA (rRNA) and transfer RNA (tRNA). Short chains of 15 thymidine monophosphates (oligodeoxythymidylic acid [oligo(dT)]) are attached to cellulose beads, and the oligo(dT)–cellulose beads are packed into a column. Total RNA extracted from eukaryotic cells or tissues is passed through the oligo(dT)–cellulose column, and the poly(A) tails of the mRNA molecules bind by base-pairing to the oligo(dT) chains. The tRNA and rRNA molecules, which lack poly(A) tails, pass through the column.
