276 7.4  Molecular Cloning

conferring resistance against a specific antibiotic that would otherwise be lethal to the cell.

For example, in bacteria, there are several resistance genes available that are effective against

broad-​spectrum antibiotics such as ampicillin, chloramphenicol, and kanamycin. Those host

cells that have successfully taken up a plasmid vector during transformation will survive cul­

turing conditions that include the appropriate antibiotic, whereas those that have not taken

up the plasmid vector will die. Using host animal cells, such as human cells, involves a similar

strategy to engineer a stable transfection such that the recombinant DNA is incorporated

ultimately into the genomic DNA using a marker gene that is encoded into the genomic DNA

conferring resistance against the antibiotic Geneticin. Unstable or transient transfection does

not utilize marker genes on the host cell genome but instead retains the recombinant DNA as

plasmids. These ultimately become diluted after multiple cell generations and so the recom­

binant DNA is lost.

7.4.2  SITE-​DIRECTED MUTAGENESIS

SDM is a molecular biology tool that uses the techniques of molecular cloning described

earlier to make controlled, spatially localized mutations to a DNA sequence, at the level of

just a few, or sometimes one, nucleotide base pairs. The types of mutations include a single

base change (point mutation), deletion or insertion, as well as multiple base pair changes. The

basic method of SDM uses a short DNA primer sequence that contains the desired mutations

and is complementary to the template DNA around the mutation site and can therefore

displace the native DNA by hybridizing with the DNA in forming stable Watson–​Crick

base pairs. This recombinant DNA is then cloned using the same procedure as described in

section 7.4.

SDM has been used in particular to generate specific cysteine point mutations. These have

been applied for bioconjugation of proteins as already discussed in the chapter and also for a

technique called cysteine scanning (or cys-​scanning) mutagenesis. In cys-​canning mutagen­

esis, multiple point mutations are made to generate several foreign cysteine sites, typically in

pairs. The purpose here is that if a pair of such nonnative cysteine amino acids is biochem­

ically detected as forming a disulfide bond in the resultant protein, then this indicates that

these native residue sites that were mutated must be within ~0.2 nm distance. In other words,

it enables 3D mapping of the location of different key residues in a protein. This was used,

for example, in determining key residues used in the rotation of the F1Fo-​ATP synthase that

generates the universal cellular fuel of ATP (see Chapter 2).

A similar SDM technique is that of alanine scanning. Here, the DNA sequence is point

mutated to replace specific amino acid residues in a protein with the amino acid alanine.

Alanine consists of just a methyl (–​CH3) substituent group and so exhibits relatively little

steric hindrance effects, as well as minimal chemical reactivity. Substituting individual native

amino acid residues with alanine, and then performing a function test on that protein, can

generate insight into the importance of specific amino acid side groups on the protein’s bio­

logical function.

7.4.3  CONTROLLING GENE EXPRESSION

There are several molecular biology tools that allow control of the level of protein expres­

sion from a gene. The ultimate control is to delete the entire gene from the genome of a

specific population of cells under investigation. These deletion mutants, also known as gene

knockouts, are often invaluable in determining the biological function of a given gene, since

the mutated cells can be subjected to a range of functionality tests and compares against the

native cell (referred to as the wild type).

A more finely tuned, reversible method to modify gene expression is to use RNA silen­

cing. RNA silencing is a natural and ubiquitous phenomenon in all eukaryote cells in which