Molecular Biotechnology. Bernard R. Glick

      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. The mRNA is removed (eluted) from the column by treatment with a buffer that breaks the A:T hydrogen bonds.

      Figure 2.10 Schematic representation of oligo(dT)-cellulose separation of polyadenylated mRNA from total cellular RNA.

      To convert mRNA to double-stranded DNA for cloning, the enzyme reverse transcriptase, encoded by certain RNA viruses (retroviruses), is used to catalyze the synthesis of complementary DNA (cDNA) from an RNA template. If the sequence of the target mRNA is known, a short (∼20 nucleotides), single-stranded DNA molecule known as an oligonucleotide primer that is complementary to a sequence at the 3′ end of the target mRNA is synthesized (Fig. 2.11A). The primer is added to a sample of purified mRNA that is extracted from eukaryotic cells known to produce the mRNA of interest. This sample of course contains all of the different mRNAs that are produced by the cell; however, the primer will specifically base-pair with its complementary sequence on the target mRNA. Not only is the primer important for targeting a specific mRNA, but also it provides an available 3′ hydroxyl group to prime the synthesis of the first cDNA strand. In the presence of the four deoxyribonucleotides, reverse transcriptase incorporates a complementary nucleotide into the growing DNA strand as determined by the sequence of the template mRNA strand. To generate a double-stranded DNA molecule, the RNA:DNA (heteroduplex) molecules are treated with RNase H, which nicks the mRNA strands, thereby providing free 3′ hydroxyl groups for initiation of DNA synthesis by DNA polymerase I. As the synthesis of the second DNA strand progresses from the 3′ ends of the nicked mRNA fragments, the 5′ exonuclease activity of DNA polymerase I removes the ribonucleotides of the mRNA. After synthesis of the second DNA strand is completed, the ends of the cDNA molecules are blunted (end repaired and polished) with T4 DNA polymerase, which removes 3′ extensions and fills in from 3′ recessed ends. The double-stranded cDNA carrying only the exon sequences encoding the eukaryotic protein can be cloned directly into a suitable vector by blunt-end ligation. Alternatively, chemically synthesized short double-stranded DNA adaptors that contain a restriction endonuclease recognition sequence can be ligated to the ends of the cDNA molecules,