The GAL System
In yeast, like almost all other cells, galactose is converted to glucose-6-phosphate by the enzymes of the Leloir pathway. Each of the Leloir pathway structural genes collectively called the GAL genes are expressed at a high level, representing 0.5-1 per cent of the total cellular mRNA St John and Davis, 1981 , but only when the cells are grown on galactose as the sole carbon source. Each of the GAL genes contains within its promoter at least one, and often multiple, binding sites for the...
The T7 Expression System
This is the RNA polymerase encoded by bacteriophage T7 is different from its E. coli counterpart. Unlike the a2 2 subunit structure of the E. coli enzyme, T7 RNA polymerase is a single-subunit enzyme that binds to distinct DNA 17 bp promoter sequences 5'-TAATACGACTCACTATA-3' found upstream of the T7 viral gene it activates. E. coli RNA polymerase does not recognize T7 promoter sequences as start sites for transcription. The overall scheme for the production of target proteins using the T7...
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denatured simply by heating. The thermal energy provided by heating a DNA sample will break the relatively weak hydrogen bonds connecting the two strand of the helix, but will not affect the covalent linkages that hold each strand together Lin and Chargaff, 1966 . The separation of the two DNA strands in a helix is accompanied by changes in the physical properties of DNA. One of these changes is the way in which DNA absorbs UV light. DNA absorbs light at a wavelength of 260 nm due to the...
Phage Display
Phage display is a powerful technique for identifying peptides or proteins that bind to other molecules. DNA encoding a specific gene or a library of cDNA fragments is cloned into the M13 bacteriophage genome in frame with gene 3 see Figure 3.15 , encoding the minor coat protein pill. This results in the expressed peptide or protein being displayed on the surface of the phage particle as a fusion to endogenous pill Smith, 1985 . Phage display thus creates a physical linkage between a library of...
Phagemids
Phagemids are plasmids that contain the f1 phage origin of replication for the production of single-stranded DNA. Phagemids are generally small plasmids so that they have the ability to accept larger DNA inserts than M13-based vectors. Phagemids were originally developed in the early 1980s, when it was found Figure 3.16. The M13 life cycle. The single-stranded M13 genome is encased by coat proteins. Bacterial infection occurs when the phage particle attaches to the E. coli pilus and the single...
DNA Purification
Many of the techniques we have discussed in this chapter are heavily reliant upon the availability of relatively large quantities of purified DNA. The isolation of nuclear material from cells is a relatively straightforward process. Lysis of Proteins separated through polyacrylamide gel Block non-specific protein binding sites and add primary antibody Wash away excess primary and add labelled secondary antibody Wash away excess primary and add labelled secondary antibody Figure 2.23. Western...
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nucleotide upstream of the initiation site of RNAI, reduces the level of the RNAI transcript and consequently results in an increase in DNA replication using RNAII as the primer. Blue-white screening - screening of this type is a special form of insertional activation that can be used during the primary selection of transformants, rather than requiring a second round of screening. It utilizes the amino-terminal portion of E. coli f-galactosidase called the a-peptide encoded by the vector in a...
Ccc Aac Cac Atc Cgc Cac Aag Atc Gtg Gag Atg Gcc Cac Cac Ggc Atc Cgg Ccc Tgc P N
GTC ATC TCG CGC CAG CTG CGC GTG TCC CAC GGC TGC GTC TCC AAG ATC CTG-3' V I S R Q L R V S H G C V S K I L 3'-AGG GTG CCG ACG CAG AGG TTC TAG-5' Primer 2 Figure 4.9. PCR to detect a pathological deletion mutation. A. The wild-type DNA sequence of the sense strand of part of exon 2 from the human Pax-3 gene. The sequence shown represents the first part of the paired box, a highly conserved element that functions as a transcription factor. Certain patients suffering from Waardenburg syndrome...
The tac Promoter
The ease with which the lac promoter can be activated the addition of IPTG to E. coli cultures makes it an attractive system for producing target proteins. However, the relative weakness of the promoter means that the target gene will not be greatly over-produced. Through the analysis of many E. coli promoters, consensus sequences for the -35 and -10 regions, to which the RNA polymerase must bind to transcribe the gene, can be determined Lisser and Margalit, 1993 . The lac promoter is weak...
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Figure 1.1. The tetranucleotide model for nucleic acid structure proposed by Levene and Simms in 1926. At the time that this model was proposed, it was thought that plant and animal nucleic acid might be different, and the differences between DNA and RNA were not fully understood other hand, containing 20 different amino acids, could provide the basis for substantial variation. In 1928, Frederick Griffith performed experiments using several different strains of the bacterium Streptococcus...
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of the transcript see Figure 1.27 . Additionally, the mRNA undergoes splicing to remove the introns so that the translation of a single contiguous message can occur. The problem with mRNA is, of course, that it cannot be maintained in stable vectors and is difficult to manipulate. Consequently, a DNA copy called complementary DNA, or cDNA of the mRNA is required before a library can be constructed. The conversion of RNA to DNA is dependent upon the action of reverse transcriptase, an enzyme...
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3'- I_I ccccC I TTTTT I 1-5' Thermostable DNA polymerase dNTP primers ' DCD PCR amplified cDNA Figure 5.7. RT-PCR to produce an amplified cDNA library sufficient prior to the progression of the PCR stage, where second-strand cDNA synthesis and subsequent PCR amplification is performed using a thermostable DNA polymerase - e.g. Taq DNA polymerase see Chapter 4 . In addition to their DNA-dependent DNA polymerase activity, some thermostable DNA polymerases e.g. Thermus thermophilus Tth DNA...
Retrovirus
Retroviruses are the only animal viruses that integrate into the host cell genome during the normal growth cycle. There are two classes of retrovirus that infect humans - the HTLV retroviruses e.g. human T-cell leukemia virus, HTLV-1 Yoshida and Seiki, 1987 , and the lentiviruses e.g. the human immunodeficiency virus, HIV-1 Luciw et al., 1984 - but retroviruses have been found in a wide variety of organisms, including both vertebrates and invertebrates. They are enveloped particles...
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Figure 10.9. The construction of plasmids producing different prey proteins for use in a genome-wide two-hybrid screen. Genes are amplified using primers that contain common sequences at their 5'-ends. This allows their re-amplification using a second pair of primers such that all genes are tagged at their 5 - and 3'-ends Hudson et al., 1997 . The tagged genes are then mixed with a linearized plasmid and transformed into yeast. Homologous recombination between the plasmid and the PCR product...
The Histag
The simplest of all protein purification tags, the his-tag is normally composed of six histidine residues. The DNA encoding these residues is cloned into the target gene such that the produced protein contains, at some point in its polypeptide sequence, six consecutive histidine residues Hoffman and Roeder, 1991 . Cloning is often performed such that the tag is located at either the extreme amino- or extreme carboxy-terminal end of the protein, where it is less likely to impair protein...
Saccharomyces cerevisiae
Baker's yeast, S. cerevisiae, is a single-celled eukaryote that grows rapidly a doubling time of approximately 90 min in simple, defined media similar to those used for E. coli cell growth. Proteins produced in S. cerevisiae contain many, but not all, of the post-translation modifications found in higher-eukaryotic cells. For example, human a-1-antitrypsin, a 52 kDa serum protein involved in the control of coagulation and fibrinolysis, is normally glycosylated. However, if the protein is...
Ebi Restriction Enz
may significantly affect the experiment. The problem of error introduction does mean, however, that PCR products should be subjected to DNA sequence analysis before they are used in cloning experiments. Additionally, several independent PCR clones should be chosen to ensure that the sequence obtained is representative. Another functional aspect of Taq DNA polymerase that impinges upon the sequence of the final PCR product is the tendency of the enzyme to incorporate a deoxynucleotide often an...
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 a-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...
M13 Vectors
M13, and its very close relatives f1 and fd, are filamentous E. coli bacterio-phages. M13 is a male-specific lysogenic phage with a circular single-stranded DNA genome 6407 bp in length Figure 3.15 . M13 phage particles have dimensions of about 900 nm x 9 nm and contain a single-stranded circular DNA molecule designated as the strand . M13 infects bacteria that harbour the F pilus. The phage particle absorbs via one end to the F pilus, and the single-stranded phage DNA enters the bacterium...
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Figure 2.6. The mechanism of DNA joining by DNA ligase. See the text for details. This figure is adapted from Doherty et al. 1996 The ability to break and rejoin DNA molecules almost at will led to the first experiments in DNA cloning in 1972 Jackson, Symons and Berg, 1972 . Figure 2.7. Breaking and joining DNA using restriction enzymes and DNA ligase. Linear DNA insert and a closed-circular plasmid DNA vector each contain the recognition site for BamHI and EcoRI. Mixing the DNA fragments with...
Pronuclear Injection
As with the methods we have previously discussed for the direct injection of DNA fragments into Xenopus oocytes Chapter 12 , DNA can be injected directly into the pronuclei of freshly fertilized mouse eggs Palmiter and Brinster, 1986 . Immediately following fertilization, the large male and small female pronuclei are visible under the microscope as discrete entities. DNA injections are usually made into the larger male pronucleus while the egg is being held in position using a suction pipette...
The Restriction Enzyme Ecorirecognizes And Cleaves Dna
Figure 2.1. Protection of DNA cleavage by modification. The restriction enzyme EcoRI recognizes and cleaves the sequence 5 -GAATTC-3'. E. coli R strains, from which this enzyme is derived, protect their own DNA from fragmentation by also producing a specific methylase. EcoRI methylase takes a methyl group from S-adenosylmethionine and places it on the N6 of the second adenine in the recognition sequence. EcoRI restriction enzyme is unable to cleave the methylated DNA semiconservative DNA...
Contents
Abbreviations and acronyms xvii 1 DNA Structure and function 1 1.1 Nucleic acid is the material of heredity 2 1.2 Structure of nucleic acids 7 1.3.1 The antiparallel helix 12 1.3.2 Base pairs and stacking 14 1.3.3 Gaining access to information with the double helix without breaking it apart 16 1.4 Reversible denaturing of DNA 18 1.5 Structure of DNA in the cell 21 1.6 The eukaryotic nucleosome 24 1.7 The replication of DNA 28 1.9 The replication process 33 1.12 Genes within a genome 40 1.13.1...
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Figure 2.13. The migration of DNA fragments through an agarose gel. a An agarose gel showing the separation of DNA fragments of known size. b The size of a DNA fragment and the distance migrated through the gel. c A plot of fragment size against distance migrated taken from the data shown in b . This indicates that the relationship between fragment size and distance migrated is not linear. d A plot of the log of fragment size against distance migrated. This indicates that there is a direct,...
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Figure 4.12. RT-PCR. The enzyme reverse transcriptase uses a complementary oligonucleotide to prime DNA synthesis from an RNA molecule. The single strand of DNA produced is then used as a template for the synthesis of a second DNA strand, and then for amplification by PCR devised as a method of RNA amplification and quantitation after its conversion to DNA. RT-PCR can be used for cloning, cDNA library construction and probe synthesis. The technique consists of two parts Figure 4.12 - the...
Genomic Libraries
The smallest unit of DNA within a genome is the chromosome. Even in the simplest organisms, however, chromosomes contain an enormous quantity of DNA. For example, the E. coli chromosome contains some 4.6 Mbp 4 600 000 bp of DNA Table 5.1 . This amount of DNA is far too large to be cloned into any of the vectors currently available Chapter 3 . Therefore it is necessary, and indeed desirable, to fragment the DNA before it is cloned into an appropriate vector. A 'divide and conquer' strategy comes...
The Eukaryotic Nucleosome
Each cell within our body contains a huge amount of DNA. The different chromosomes of the human genome contain approximately 3.2 x 109 base pairs of DNA. Since we are diploid organisms, having two sets of each chromosome, the total amount of DNA in most of our cells totals 6.4 x 109 base pairs. At 0.33 nm per base pair Figure 1.7 , this corresponds to an overall length of approximately 2.1 m. How can this fit into a nucleus measuring just 5-10 m across The answer is that the DNA is highly...
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Figure 5.10. Enrichment of DNA molecules during subtractive hybridization. The subtraction of the driver library from the target library results in the elimination of common sequences genes 1 and 2 and the enrichment of sequences that are more abundant in the target library genes 3-5 . Shown here are agarose gels of the libraries stained with ethidium bromide EtBr , and then blotted and probed with the DNA sequences for five separate genes. The intensity of the band in the probed gels indicates...