John Wiley & Sons - 2004 - Analysis of Genes and Genomes

a functional transcriptional activator protein within the promoter of a reporter gene, whose expression can then be detected (Figure 6.12).

6.7.1One Hybrid

In a simplification of the two-hybrid system, a cDNA –AD library is introduced into a yeast strain that harbours a reporter gene into which DNA binding sites have been introduced. Proteins encoded by the cDNA that are able to bind to the DNA binding sites will activate the transcription of the reporter gene (Li and Herskowitz, 1993). This approach is useful in the identification of proteins that regulate the promoters of known genes (Wang and Reed, 1993). To perform a screen like this, the DNA binding element is usually multimerized to produce a strong activation element (through the binding of multiple activator proteins) so that expression of the reporter can be observed easily.

6.7.2Three Hybrid

Any mechanism by which the DBD and AD functions of the transcriptional activator may be brought together can be used to activate the reporter gene. Several screens have been described in which the DBD and AD fusion proteins do not interact directly with each other, but their interaction is mediated through another factor. For example, SenGupta et al. connected two RNA binding proteins (one fused to the DBD and one fused to the AD) through a bifunctional RNA molecule produced within the yeast cells (SenGupta et al., 1996). Screens could then be established to look for RNA binding proteins that bind novel sequences incorporated into the RNA linker. In a similar vein, Licitra and Liu developed a system to detect small-molecule–protein interactions (Licitra and Liu, 1996). In this system, the DBD was fused to a ligand binding protein (a receptor than binds the hormone dexamethasone) and a bifunctional ligand (containing dexamethasone and other groups) was used to screen for proteins in a cDNA –AD fusion library that bound to the other parts of the bifunctional ligand. In both the cases described here a non-protein component holds the DBD and AD together to allow transcription to occur.

6.7.3Reverse Two Hybrid

The interaction between the bait and the prey is used to drive the expression of a gene whose product is lethal to the cell (Leanna and Hannink, 1996). This is useful to screen for drugs that disrupt the interaction between the proteins and thereby allow the cells to survive through the non-expression of the reporter (Huang and Schreiber, 1997).

232 CREATING MUTATIONS 7

single-cell eukaryotes with X-rays, UV light or chemicals such as ethyl methane sulphonate (EMS) generates DNA base changes throughout the genome. Once produced, these changes will be passed from generation to generation as the cells divide. This type of traditional mutagenesis has, however, a number of drawbacks. For example, the mutations produced are random – they can occur anywhere within a genome and are not restricted to individual genes or parts of genes. Additionally, highly developed and specialized screening procedures are required to identify mutations that have occurred within individual genes. This is relatively straightforward for mutations occurring in genes that encode, for example, one of the enzymes of a metabolic pathway. Mutations that destroy the activity of one member of the pathway are likely to lead to the formation of an organism that is unable to metabolize a particular nutrient. Screens based on growth assays can then be devised to isolate these mutants. Traditional forms of mutagenesis also suffer, since the observed phenotypic change in a screen may not be a result of a mutation within a single gene. Additionally, multiple mutations may be required (perhaps when multiple redundant genes occur within the same cell) before a phenotypic change can be observed.

Mutations within DNA generally fall into one of two categories. In the first, a base or bases within a DNA sequence are changed from one sequence to another, while in the second bases are either inserted into or removed form the gene. Single DNA base pair changes are described as being either transition mutations or transversion mutations:

•transition mutations – the change of one purine –pyrimidine base pair to a different purine –pyrimidine base pair (e.g. AT → GC, or GC → AT, or TA → CG);

•transversion mutations – the change of a purine – pyrimidine base pair to a pyrimidine –purine base pair (e.g. AT → TA, or GC → CG, or AT → CG, or GC → TA).

Single base changes may result in various alterations to the amino acid sequence of the protein encoded by the gene at the regions of the changed bases. The mutation may be a

•silent mutation – the triplet code is changed, but the amino acid encoded is the same (e.g. the triplets 5 -TCG-3 and 5 -TCC-3 both encode the amino acid serine),

•mis-sense mutation – a codon change alters the amino acid encoded (e.g. if the serine codon 5 -TCG-3 is mutated to 5 -ACG-3 , then the amino acid threonine will be inserted into the encoded polypeptide in place of serine – or

234 CREATING MUTATIONS 7

double-stranded form. That is, the single-stranded form of the genome serves as a template for the new synthesis of a second DNA strand. This process can be used to our advantage if we want to create mutations within the newly synthesized DNA strand, and is outlined in Figure 7.1. The use of oligonucleotides in creating site-directed mutations was devised in the laboratory of Michael Smith, who shared the 1993 Nobel Prize in Chemistry for his discovery. Smith and his colleagues used single-stranded M13 genomic DNA as a hybridization template for a synthetic oligonucleotide (Zoller and Smith, 1983). The oligonucleotide binds to its complementary sequence within the single stranded genome, and is designed such that one or more mutations (non-complementary base pairings) occur when it binds to the M13 DNA. The binding of the oligonucleotide to the single-stranded DNA is stabilized by the complementary base pairing that occurs elsewhere. In addition to altering individual bases, an oligonucleotide can also introduce base insertions or deletions into a gene. Once bound to its complementary sequence, the oligonucleotide provides a free 3 hydroxyl group as the starting point of DNA synthesis. The hybrid, partially double-stranded, DNA molecule is incubated with a DNA polymerase enzyme in the presence of the four deoxynucleotide triphosphates (dNTP). This will result in the synthesis of a new DNA strand that is entirely complementary to the original DNA strand except at the positions where mutations have been introduced within the oligonucleotide itself. The newly synthesized DNA circle is then completed by the action of DNA ligase, in the presence of ATP, to seal any nicks remaining in the DNA backbone. The naked DNA is unable to infect E. coli cells, so it must be introduced into the bacterium where the DNA will be replicated and phage particles produced. When the DNA circles are replicated in bacterial cells, one of two possibilities can arise – either the original wild-type DNA strand or the newly synthesized mutated DNA strand can give rise to progeny M13 bacteriophages. That is, the resulting M13 plaques may either contain the wild-type sequence or the mutated sequence.

Bacteriophages containing either the wild-type or the mutant sequence can be distinguished from each other through hybridization screening (similar to that described in Chapter 6). A radio-labelled version of the synthetic oligonucleotide used to create the mutation will bind preferentially to the mutant sequence when compared with the wild-type sequence (Wallace et al., 1981). Therefore, bacteriophage plaques that are able to bind the oligonucleotide at high stringency should contain the mutant sequence.

The primer extension site-directed mutagenesis procedure became widely adopted in the early 1980s. It suffered, however, from a number of drawbacks as a method for rapidly producing a variety of specific DNA mutations.

in which the mutation frequency approaches 100 per cent would mean that only a single bacteriophage plaque (or possibly two) would need to be analysed to ensure that a mutant can be isolated. Lower mutation frequencies result in a greater number of phages that need to be analysed before a mutant is likely to be found, and has consequences for the speed at which specific mutations may be isolated. Methods have been devised to increase the overall efficiency of a mutagenesis experiment by either increasing the efficiency of the mutagenesis reaction itself, or by the use of bacterial strains that are less likely to degrade the newly formed mutant DNA strands. For example, E. coli cells that are defective in the mutL, mutS, mutH mis-match repair system can be used for the transformation of the hybrid DNA molecules so that the mutation cannot be repaired back to the wild-type sequence.

7.2Strand Selection Methods

An extremely effective approach to increasing the mutagenesis efficiency is to devise procedures to select either for the mutant DNA strand, or against the wild-type DNA strand. Here, we will only describe two methods for the latter that remain in use today. In the first the incorporation of nucleotide analogues protects the newly synthesized mutant DNA strand from degradation in vitro (Taylor, Ott and Eckstein, 1985), while in the second the wild-type DNA strand is targeted for degradation within E. coli cells (Kunkel, 1985).

7.2.1Phosphorothioate Strand Selection

A phosphorothioate nucleotide contains a phosphorus –sulphur linkage in place of a phosphorus –oxygen group (Figure 7.3). If phosphorothioate deoxynucleotides in which the sulphur is attached to the α-phosphate are used in a DNA synthesis reaction, then the phosphorothioate will be incorporated into the newly synthesized DNA. Certain restriction enzymes are unable to cleave DNA that contains phosphorothioates (Nakamaye and Eckstein, 1986). The mutagenic oligonucleotide is annealed to the single-stranded M13 DNA template as described above, but is extended by DNA polymerase in the presence of three deoxynucleotide triphosphates (dATP, dTTT and dGTP) and a single phosphorothioate nucleotide (dCTPαS). This will result in the formation of the newly synthesized mutant DNA strand, but not the wild-type strand, containing a phosphorothioate at every C residue. The cleavage of the DNA duplex with, for example, the restriction enzyme PstI will result in the nicking of the wildtype DNA strand (no phosphorothioate) but no cleavage of the mutant DNA