analysis of genes and genomes phần 6 pps

228 GENE IDENTIFICATION 6 domain of the Herpes simplex virus protein VP16 or the artificial activating sequence B42), reviewed by Brent and Finley (1997). The principles described above for the Gal4p based systems hold true for each of these modified versions. 6.7 Other Interaction Screens – Variations on a Theme The two-hybrid screen has been modified extensively to suit different purposes. The basic objective behind each type of screen that is listed below is to assemble Gal4p AD LexA DBD AD RNA binding protein 1 Gal4p DBD AD cDNA DBD Multimerised binding sites One-hybrid screen to identify DNA-binding domains (a) Reporter gene Reporter gene Reporter gene Reporter gene Reporter gene Reporter gene Bifunctional RNA RNA binding protein 2 UAS G Three-hybrid screen to identify RNA-binding proteins Three-hybrid screen to identify ligand-binding proteins Bifunctional ligand Ligand binding protein 1 Ligand binding protein 2 LexOp (b) (c) Figure 6.12. Other types of interaction screening. (a) One-hybrid screening. (b), (c) Three-hybrid screening. See the text for details 6.7 OTHER INTERACTION SCREENS – VARIATIONS ON A THEME 229 a functional transcriptional activator protein within the promoter of a reporter gene, whose expression can then be detected (Figure 6.12). 6.7.1 One 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. 6.7.2 Three 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. 6.7.3 Reverse 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). 7 Creating mutations Key concepts  Mutations – the altering of one DNA sequence to another – are vital to our understanding of how genes and proteins function  DNA sequences can be altered in a specific, highly directed way  Oligonucleotide binding to complementary DNA sequences can be used to create mutant mis-matches. The mutant oligonucleotide is then used as a primer for DNA synthesis such that the new DNA contains mutations  Strand selection methods distinguish between the newly synthesized mutant DNA and the parental wild-type sequence  PCR can be used to create either specific or random mutations The sequence of a gene dictates the amino acid sequence of the protein that it encodes. Consequently, mutations within a gene, i.e. alterations to the DNA sequence, may result in changes to the amino acid sequence of the protein. The analysis of mutations is especially useful in the elucidation of protein function. Mutations that either reduce the activity of the protein, or allow the protein to behave in an abnormal way, can be used to ascribe particular functions to individual portions of proteins. The rate of naturally occurring mutations, resulting as a consequence of erroneous DNA replication within genes, is quite low (estimated at a level of one DNA base alteration per 10 6 –10 8 bases replicated) and varies widely between individual genes and organisms. Naturally occurring mutations have, however, been used to isolate genes and describe specific functions for their encoded proteins. Prior to the explosion in molecular biology techniques in the 1970s and 1980s, increased mutation rates were usually obtained by treating whole cells with either a physical or a chemical mutagen. For example, the treatment of micro-organisms or Analysis of Genes and Genomes Richard J. Reece  2004 John Wiley & Sons, Ltd ISBNs: 0-470-84379-9 (HB); 0-470-84380-2 (PB) 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 theserinecodon5  -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 7.1 CREATING SPECIFIC DNA CHANGES USING PRIMER EXTENSION MUTAGENESIS 233 • non-sense mutation – an amino acid codon is changed to produce a stop codon. For example, if the serine codon 5  -TCG-3  is mutated to 5  -TAG-3  , then the encoded polypeptide chain will terminate at this point. The insertion or deletion of a base pair, or base pairs, into the coding sequence of a gene can have drastic implications for the encoded polypeptide. Since the DNA code is read in triplets, the insertion or deletion of bases in multiples other than three will result in a frame-shift mutation. If, for example, a single letter in the sentence below is deleted, then the meaning of the sentence beyond the change is radically altered: THE FAT CAT ATE THE RAT THE FAT CTA TET HER AT. Deletion of A Similarly, the deletion, or insertion, of one or two bases into the coding sequence of a gene will muddle the remainder of the sequence beyond the mutation. Only the deletion or insertion of multiples of three bases will leave the remainder of the encoded polypeptide sequences unaltered, but will remove (or insert) amino acids. THE FAT CAT ATE THE RAT THE CAT ATE THE RAT Deletion of FAT The alteration of a single amino acid to another within a protein can have great consequences on the function of the protein itself. To enable a highly directed approach to the study of the relationship between genes and proteins, mechanisms to create mutations at highly specific regions of DNA are required. 7.1 Creating Specific DNA Changes Using Primer Extension Mutagenesis As we have already seen in Chapter 3, bacteriophage M13 undergoes a switch during its life cycle during which its single-stranded genome is converted to a 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 oligonu- cleotides in creating site-directed mutations was devised in the laboratory of Michael Smith, who shared the 1993 Nobel Prize in Chemistry for his dis- covery. 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 indi- vidual 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 oligonu- cleotide 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. 7.1 CREATING SPECIFIC DNA CHANGES USING PRIMER EXTENSION MUTAGENESIS 235 C G T A C A T 5 ′ - G C A G G T A - 3 ′ C G T A C A T Add oligonucleotide 5′-GCAGGTA-3′ Single- stranded M13 C G T A C A T G C A G G T A C G T A C A T G C A T G T A C G T C C A T G C A G G T A 1. DNA polymerase + dNTP 2. T4 DNA ligase + AT P Transform into E. coli or Wild-type Mutant Figure 7.1. Site-direct mutagenesis using a single-stranded DNA template. Single- stranded DNA isolated from a recombinant M13 phage bearing the gene to be mutated is isolated and used as a template for the binding of an oligonucleotide primer. The primer hybridizes to its complementary sequence and introduces a specific mutation(s). The hybrid is then treated with DNA polymerase in the presence of the four deoxynucleotide triphosphates (dNTP) to synthesize a new M13 DNA strand complementary to the original at every base, except for those alterations introduced in the primer. The sugar–phosphate backbone of the new DNA circle is then completed using DNA ligase and the double- stranded DNA is transformed into E. coli cells. In E. coli , either the newly synthesized mutant DNA strand or the original wild-type DNA strand will be used to create new M13 phages 236 CREATING MUTATIONS 7 • The DNA that is to be mutated needs to be cloned into the M13 genome. • The efficiency of the mutagenesis procedure itself is quite low. In order to create a newly synthesized mutant DNA strand, efficient oligonucleotide binding, DNA replication and DNA ligation are required. Each of these procedures is likely to be less than 100 per cent efficient, and therefore wild-type DNA strands will predominate in the mixture that is transformed into bacteria. • The newly synthesized DNA will not be methylated as it is produced in vitro, while the wild-type M13 genome, isolated from bacterial cultures, will be methylated. This is important because the mismatch repair systems of the E. coli favour the repair of non-methylated DNA (Kramer and Fritz, 1984). This will result in the mismatches between wild-type and the mutant DNA strands being repaired in favour of a return to the wild-type sequence. • The differential screening procedure to identify mutant phages is both slow and cumbersome and often results in the isolation of wild-type rather than mutant phage. All of these factors result in a mutation efficiency that is low. Typically, mutage- nesis frequencies of less than 10 per cent might be obtained for primer extension reactions like those described above. A consequence of the low mutation fre- quency is that a large number of potential mutants need to be screened to ensure that at least one genuine mutant can be isolated (Figure 7.2). Procedures 0 102030405060708090100 Efficiency of mutation (%) 0 5 10 15 20 Number of clones analysed to give 90% chance of isolating the mutant Figure 7.2. Efficient mutagenesis procedures greatly reduced the number of clones that must be screened to a single specific mutant 7.2 STRAND SELECTION METHODS 237 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. 7.2 Strand 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). 7.2.1 Phosphorothioate Strand Selection A phosphorothioate nucleotide contains a phosphorus–sulphur linkage in place of a phosphorus–oxygen group (Figure 7.3). If phosphorothioate deoxynu- cleotides 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 tem- plate 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 wild- type DNA strand (no phosphorothioate) but no cleavage of the mutant DNA [...]... If the sense strand of PCR 2 binds to the antisense strand of PCR 1, then a molecule is produced that cannot be extended using DNA polymerase (the 3 -ends are not base paired) However, if the sense strand of PCR 1 binds to the antisense strand of PCR 2, then DNA polymerase can produce a double-stranded version of the gene that contains the mutation In practice, the products of PCR 1 and PCR 2 are mixed... RBS Protein Figure 8.2 DNA sequences of the lac, trp and tac promoters The consensus E coli −35 and −10 sequences based on the analysis of naturally occurring promoters are shown above, and the sequences of each of the promoters, extending from the −35 region to the translational start site, are shown The tac promoter is a hybrid of the trp and lac promoters The −35 and −10 regions it contains closely... mutation and sequences at the 3 -end of the sense strand The two PCR products formed by this process can anneal to each other through their complementary sequences, as dictated by the position of primers 2 and 3 The mixed DNA strands can then be amplified using primers 1 and 4 to generate the intact DNA fragment that now contains the mutation 7.4 PCR BASED MUTAGENESIS 243 to the anti-sense strand and the... strand and the sense strand of the target DNA, respectively The other two primers (2 and 3) are designed to bind to the different strands of the same DNA sequence and will also introduce the required mutation into each strand In the first PCR, the 5 -end of the gene is amplified using primers 1 and 2 The resulting product will bear the mutation at its 3 -end In the second PCR, the 3 -end of the gene is amplified... to the physical size of a cassette that can be produced effectively We will return to cassette mutagenesis again when we look at the production of random mutations in specific genes 7.4 PCR Based Mutagenesis We have already seen how, by suitable design of oligonucleotide primers, mutations can be introduced into the ends of PCR products in a way that leads to mutagenesis efficiency of almost 100 per cent... MUTAGENESIS (a) Gal4 binding site Put3 binding site 247 Ppr1 binding site DNA-protein complexes Free DNA - 1 2 3 4 5 6 7 8 9 - 1 2 3 4 5 6 7 8 9 Zinc cluster Linker Dimerization (b) - 1 2 3 4 5 6 7 8 9 Binding Specificity Protein 1 Zn Gal4p(1−100) GAL4 2 Zn Gal4p(1−38)+ Put3p (61 −1 26) PUT3 3 Zn Gal4p(1 61 )+ Put3p(84−1 26) GAL4 4 Zn Put3p(31 60 )+ Gal4p(39−100) GAL4 5 Zn Put3p(31−79)+ Gal4p(58−100) PUT3 6. .. sequence are likely located on the surface of the protein and may therefore participate in, for example, protein–protein interactions Mutation of these charged clusters are more likely to disrupt these protein–protein interactions than mutagenesis of other residues Two approaches are commonly used for the creation of random mutations within individual genes, or parts of genes Again, these methods rely on a... expression of any gene placed under its control through the recruitment of RNA polymerase to that gene Much work has gone into the design of vectors for maximizing protein production The architecture of a typical expression vector is shown in Figure 8.1 Analysis of Genes and Genomes Richard J Reece  2004 John Wiley & Sons, Ltd ISBNs: 0-470-84379-9 (HB); 0-470-84380-2 (PB) 258 PROTEIN PRODUCTION AND PURIFICATION... the ends of linear DNA fragments, but is limited to those ends PCR protocols have, however, been developed to enable the creation of mutation at any point throughout the length of the PCR product (Higuchi, Krummel and Saiki, 1988) This method, often referred to as two-step PCR mutagenesis, requires four oligonucleotide primers and three separate PCR reactions and is outlined in Figure 7 .6 Two of the... −35 region of the E coli trp operon, and the −10 region of the lac operon, controlling the expression of the genes responsible for tryptophan biosynthesis and lactose metabolism, respectively, results in the formation of the tac promoter, which is five times as strong as the lac promoter itself (de Boer, Comstock and Vasser, 1983) The tac promoter is able to induce the expression of target genes such . the anti-sense strand and the sense strand of the target DNA, respectively. The other two primers (2 and 3) are designed to bind to the different strands of the same DNA sequence and will also introduce. Krummel and Saiki, 1988). This method, often referred to as two-step PCR mutagenesis, requires four oligonucleotide primers and three separate PCR reactions and is outlined in Figure 7 .6. Two of the. strand of PCR 1 binds to the antisense strand of PCR 2, then DNA polymerase can produce a double-stranded version of the gene that contains the mutation. In practice, the products of PCR 1 and PCR