ANÁLISIS DE MALLAS

2.1.1 Introduction

In this thesis several powerful molecular biology techniques are used throughout. Before the early 1970s, DNA was an extremely difficult molecule to analyse due to its length and chemical monotony. Molecular biology was revolutionised by the discovery o f enzymes that can cut, join and replicate DNA, and also reverse transcribe RNA. Prokaryotic restriction enzymes, discovered by Wemer Arber in 1962, recognise specific base-pair sequences in double stranded DNA and cut both strands at specific sites. This allows cleavage of DNA molecules into smaller fragments that are more easily analysed and manipulated. A second major advance was the finding that denatured, single-stranded DNA and RNA molecules will readily reform double-stranded molecules with complementary nucleotide sequences at 65°C. This process, called DNA hybridisation, can occur between any two complementary single-stranded nucleic acid chains (DNA/DNA, DNA/RNA, RNA/RNA). Additionally, in the early 1970s methods were developed to determine the nucleotide sequence o f any purified DNA fragment, making it possible to determine the complete DNA sequences of thousands o f genes and, eventually, the entire human genome. A powerful technique known as the Polymerase Chain Reaction (PGR) was also introduced in the 1970s with the discovery o f a heat-

amplification of a particular sequence of DNA (providing that at least part o f the sequence is known). Finally, advances in microbial genetics found application in molecular biology, such as the use of viral enzymes to reverse transcribe mRNA to complementary DNA and the extensive use o f bacterial plasmids to package and deliver recombinant DNA molecules into host cells.

Plasmids are circular double-stranded DNA molecules that are found in many bacterial species. They act as accessory chromosomes that can replicate themselves and drive mRNA transcription independently o f the host genome. However they require the transcription and translation machinery o f the host cell. Such plasmids have been cleverly modified to allow them to act as vectors for the efficient delivery o f foreign genetic material into either bacterial or eukaryokic cells. Many plasmid vectors are now commercially available, such as the pcDNA3.1 family o f vectors used in this thesis, the main features o f which are shown in Figure 2.1. pcDNAS.l vectors are designed for high level stable or transient expression o f the encoded gene in mammalian cells, and efficient propagation of the plasmid vector in many strains o f E. coli.

Figure 2.1: The major features of the pcDNA3.1 plasmid vector family.

The pcDNA3.1 (+) vector, used in these studies for the stable expression of the Kir 3.1/3.2A channel subunits, has enhanced promoter regions from the immediate early gene of the human cytomegalovirus (CMV) to ensure high levels of expression; a multiple cloning site (MCS) containing DNA sequences that are recognised and cleaved by the restriction enzymes listed above; an Ampicillin resistance gene for selection and propagation of this vector in E. coli; and a Neomycin resistance gene which allows selection of mammalian stable lines with the antibiotic Geneticin (0418). The pcDNA3.1/zeo vector has a similar list of features to pcDNA3.1 (+) but it incorporates the Zeocin resistance gene, instead of the Neomycin resistance gene, which allows for selection of mammalian stable lines using the antibiotic Zeocin. This vector was used to establish stable cell lines expressing each of the Gj/o-coupled receptors and the Gjai-Ai receptor fused construct. Both (+) and (-) pcDNA3.1

2.1.2 Restriction Digestion of DNA molecules

Type n restriction enzymes have been used in this thesis to: i) clone the DNA o f interest from one vector to another

ii) linearise double-stranded plasmid DNA to confirm its identity

Type n restriction enzymes cleave DNA at specific nucleotide sequences. Many restriction enzyme sites contain a two-fold axis o f symmetry; some enzymes cleave at the axis o f symmetry producing blunt ends while other restriction enzymes (eg. EcoRl) cut in a staggered fashion on either side o f the axis. The staggered cuts made by EcoRl produce complementary single-stranded overhangs that have a specific affinity for each other and hence are known as cohesive ends. For DNA cloning, the plasmid DNA and the “insert” DNA are cut with the same restriction enzyme(s) giving compatible cohesive or blunt ends. The two molecules are then joined (ligated) together using the enzyme DNA ligase.

2.1.3 Agarose gel electrophoresis

Electrophoresis describes the technique whereby proteins or other macromolecules such as DNA or RNA move in an electrical field according to their net charge, size and shape. DNA fragments can be readily separated from each other on the basis of size by electrophoresis due to the uniform negative charge on DNA molecules at neutral pH. Polyacrylamide gels are suitable for the separation o f small nucleic acid strands, such as during DNA sequencing, whereas agarose gels have a larger pore size which is suitable for the separation of larger DNA fragments as used in this

thesis. The migration rate of linearised, double-stranded DNA molecules through an agarose gel is proportional to the logarithm o f the number o f nucleic acid base pairs (Helling et al., 1974). Different conformations o f DNA (i.e. supercoiled circular, nicked circular) migrate at different rates. In my studies DNA fragments that were applied to agarose gels were linearised by digestion with restriction enzymes. Occasionally non-linearised supercoiled DNA was applied to a gel in order to approximate the concentration o f DNA in the sample.

To visualise DNA bands, the fluorescent intercalating dye ethidium bromide was added to the agarose gel. The horizontal gel slab configuration was used in these studies. 0.7 to 1.0% agarose gels were used to resolve DNA fragments. Agarose gels were prepared by dissolving 0.7g o f agarose (for a 0.7% gel) in 100ml o f IX Tris Acetate EDTA (TAB; made up from a 5OX stock solution in distilled water) by superheating the solution in a microwave at full power for 3-5 minutes. The solution was allowed to cool prior to adding lOpI (lOOpg) o f the DNA-binding dye, ethidium bromide, supplied in aqueous solution (lOmg per ml) (Sigma, Poole, UK). The gel solution was poured promptly into a plastic tray with tape applied to seal both ends, and plastic combs inserted so as to create 20-3Opl volume wells.

Prior to running, a gel was allowed at least 1 hour to set, and tape was removed from the ends o f the gel tray. The agarose gel was placed in a Horizontal Gel Electrophoresis tank (Horizon 11.14; Gibco Life Technologies, Paisley, UK) that was filled with fresh IX TAE buffer so as to cover the gel and to fill the wells on

(usually the 0.37-8.0kbp DNA Molecular Weight marker; Roche Diagnostics, Mannheim, Gennany) was also prepared by adding DLB to ladder in a similar ratio (ie. Ipl DLB to 5pi ladder). Samples were applied to the gel by pipetting into appropriate wells and an electrophoresis voltage o f 60-100V was applied across the electrodes. Migration distance was judged by movement o f the tracker dyes, bromophenol blue and xylene cyanol.

Once the samples had migrated an appropriate distance, the gel was viewed under ultra-violet (UV) light (312nm wavelength) using a UVP dual intensity transilluminator (Upland, California, USA) equipped with a digital video camera. The transilluminator/camera was linked to a black and white monitor (Sony SSM- 12ICE) and photographs o f the gel were printed by a video graphic printer (Sony UP-890CE).

L 4

7 8 9 10 G L

Figure 2.2: Digest of cDNA obtained from the Guthrie cDNA Resource Centre. All Guthrie RGS cDNAs had been subcloned into pcDNA3.1(+) (Invitrogen) at the Kpnl (5’) and Xhol (3’) sites and were dispatched as desiccated samples on blotting paper. In our lab, each individual DNA sample was redissolved in distilled water, transformed in to supercompetent E. coli and plated onto agar plates containing lOOpg/ml carbenicillin. Discrete colonies were picked and grown in 30ml LB broth with lOOpg/ml carbenicillin, and then plasmid DNA was purified from this bacterial culture using a QIAGEN plasmid Midi kit (QIAGEN, Crawley, UK). To confirm the identity of the purified DNA, 5pi of each sample was digested with Kpnl and Xhol to excise the RGS cDNA insert of known size. Lane 2 - Lane 7 show the digested products of RGS4, 7, 8,9, 10 and RGS-GAIP (G). Lanes I and 8 contain lOObp and 0.37-8.Okbp DNA Molecular Weight markers respectively (Roche Diagnostics, Mannheim, Germany). The molecular weights of the excised DNA fragments corresponded to the sizes of the RGS cDNA inserts: 550bp for RGS4, I330bp for RGS7, 550bp for RGS8, I350bp for RGS9 and 5I0bp for RGS 10. The expected 660bp excised fragment from RGS-GAIP was absent on this gel indicating that the plasmid DNA had lost the insert DNA for RGS-GAIP. A new sample of RGS-GAIP

Co-migration of the DNA Molecular Weight markers allowed calibration o f the size o f bands appearing on the DNA gel, and visual comparisons o f the intensity o f the bands with that o f the DNA standards gave an estimation o f the DNA concentration in each sample. In this thesis, DNA agarose gels were used:

1) diagnostically, to confirm the identity o f new or subcloned cDNAs (Fig. 2.2), 2) to confirm the purity o f a DNA preparation and estimate its concentration,

3) to separate specific DNA fragments from a mixture o f digested fragments following a restriction enzyme digest. The separated DNA fragment can be excised and purified from the agarose gel as described in the next section.

2.1.4 Excision and purification of DNA fragments from agarose gels.

A commercially available kit was used in this thesis to extract and purify DNA from agarose gels. The QIAEX gel extraction kit (Qiagen, Crawley, Sussex, UK) first solubilises the agarose and then selectively absorbs the DNA onto a resin o f silica- gel particles under high salt conditions. Contaminants are removed by a series of washes and then the DNA is eluted from the resin with distilled water. The details o f the procedure followed are given below.

The DNA fragment of interest was identified on the agarose gel under UV light, excised using a clean scalpel and placed in a pre-weighed eppendorf tube. The weight o f the gel slice was recorded and this was used to calculate the volume o f QXl buffer required to solubilise the agarose. QXl buffer has a high concentration of chaotropic salts that disrupt the structure of water and thereby promote the

solubility of nonpolar substances in polar solvents. DNA fragments less than 4kb in size required 3 volumes o f QXl buffer per 1 volume of gel slice for solubilisation (for fragments greater than 4kb in size 3 volumes o f QXl and 2 volumes o f distilled water was used). The agarose was solubilised by incubation with QXl buffer at 50°C for 10-15 minutes. Then lOpl o f QIAEX II resin was added to bind the DNA. Binding was allowed to proceed for 10 minutes at 50°C with regular vortexing to keep the resin in suspension. The resin was then pelleted at maximum speed in a microcentrifuge for 30 seconds, and the supernatant discarded. The pellet was washed by resuspension and subsequent pelleting with SOOpl QXl followed by two additional washes in 500pl PE buffer (a low salt buffer containing ethanol) to remove any residual salt contamination. The pellet was then air-dried for 20-30 minutes at room temperature, followed by the addition o f 2 0pl o f distilled water to elute the DNA. The pellet was resuspended by vortexing and incubated at room temperature for 5 minutes (DNA fragments greater than 4kb in size were incubated at 50°C for 5 min). Finally the resin was pelleted by centrifugation and the eluate was transferred to a clean eppendorf. To increase the yield o f DNA the elution process was repeated and the eluates combined. In general, the above extraction procedure was used to produce purified DNA for the use in ligation reactions (described below).

2.1.5 Ligation of DNA fragments

DNA fragment with the same enzyme or pair of restriction enzymes produces compatible cohesive termini. These termini can anneal to one another, and the enzyme DNA ligase can then reseal the strands by creating a phosphodiester bond.

It is relatively straightforward to ligate a cDNA fragment with its target vector when both have been digested with the same restriction enzymes that produce staggered ends. Complications arise when there are not appropriate sites for staggered-cutting restriction enzymes. In these situations it may be necessary to use blunt-cutting enzymes or even staggered-cutting enzymes that produce incompatible cohesive ends; ligation of such restriction enzyme digests is clearly more complicated. However, in all instances described in this thesis the same enzyme or pair o f enzymes was used to cut both the vector and to excise the DNA fragment o f interest, producing staggered, compatible cohesive termini.

DNA ligase catalyses the formation o f a new phosphodiester bond between phosphate residues located at the 5’ terminus o f the DNA fragment and hydroxyl groups at the 3’ end. To prevent the re-ligation of the termini o f a digested vector, the 5’ phosphate groups can be removed by the enzyme calf intestinal phosphatase (CIP). This step is particularly important when a single staggered-cutting enzyme is used producing identical cohesive ends that are highly likely to religate. However, in our laboratory, digested vectors are routinely treated with CIP because it prevents the religation o f partially digested vector and so reduces the frequency o f colonies appearing on control plates (see below).

Following the digestion o f the vector with the appropriate restriction enzymes, 10 units o f CIP (New England Biolabs, Hitchin, UK) was added to the digest reaction and incubated for a further 1 hour at 37°C. O f note, treatment with GIF leads to the formation o f single-stranded nicks in successfully ligated DNA. The absence o f 5’ phosphate groups on the vector DNA means that phosphodiester linkages will not form with the hydroxyl groups on the 3’ termini o f the inserted DNA fragment. However the nicked DNA can still be transformed into bacteria where the nicks are sealed once in the host cell (Sambrook et al. 1989).

Two types o f DNA ligase are commercially available, E. Coli ligase and

bacteriophage T4 DNA ligase. The latter enzyme is used in our laboratory because it is more efficient at ligating blunt-ended termini. T4 DNA ligase (Roche Molecular Biochemicals, Mannheim, Germany) requires Mg^^ and ATP as cofactors. These are included in the lOX ligation buffer supplied by the manufacturers that contains 660mM TrisHCl, 50mM MgCb, lOmM dithiothreitol and lOmM ATP, pH 7.5. Digested DNA fragments and vectors that had been separated by agarose gel electrophoresis and purified as described form the substrates for ligation reactions. Ligation reactions were set up in parallel with an appropriate control to monitor re­ ligation o f the CIP-treated vector. The components o f a typical set o f ligation reactions is given below:

Ligation Reaction Control Reaction

5 pi digested vector (CIP-treated) 5 pi digested vector (CIP-treated)

Ligation reactions were incubated at 16°C overnight. The following day, ligated DNA was introduced into competent E. coli by the process o f transformation as

described in the next section. Transformed E. coli was spread on agar plates

containing the appropriate antibiotic resistance to select for the target vector. For DNA constructs ligated into vectors pcDNA3.0 and pcDNA3.1 (zeo) (both from Invitrogen, Paisley, UK), lOOpg/ml carbenicillin was used to select for E. coli

transformed with the ligated DNA construct. For the vectors pEGFP-Nl, pECFP-Nl and pEYFP-Nl (Clontech, California, USA), kanamycin resistance (30pg/ml) was used to select for successfully transformed bacteria.

2.1.6 Preparation of competent E. coli for transformation with DNA

The process o f introducing foreign plasmid DNA into bacteria is known as transformation. There are two main methods o f introducing foreign DNA into bacteria, chemical transformation and electroporation, and both require pretreatment of the bacteria with specific salt buffers to render them “competent”. Competent bacteria more readily take up foreign DNA. Prior to electroporation, bacteria are washed in a series of low salt buffers. An electric pulse permeabilises the bacterial cell membrane and permits entry o f DNA. For practical reasons, chemical transformation has been used in this thesis to introduce foreign DNA into E. coli

Competent bacteria are available commercially and our laboratory purchased a strain o f E. coli known as “Top 10”, supplied in a glycerol stock (Invitrogen, Groningen,

Netherlands). Periodically a new batch o f competent bacteria would be prepared from this “master stock”, kept in storage at -80°C. Top 10 cells from the glycerol stock were streaked onto an agar plate prepared using Luria-Bertani (LB) broth.

LB Broth contains (grams per litre): 10 Tryptone, 5 Yeast Extract, 5 NaCl, and is supplied in tablet form by Sigma Aldrich (Poole, UK). Plates were incubated at 37°C for 12-16 hours and a single colony was used to inoculate 2.5ml o f LB broth; this was incubated at 37°C in a shaking incubator set at 225rpm for 12-16 hours. This starter culture was then diluted 1:400 in 200ml LB broth and incubated at 37°C, with vigorous shaking, until it reached log phase growth. The growth o f the culture was monitored by the removal of small aliquots o f culture and measurement o f its absorbance at 600nm. The culture was judged to have reached log phase when the ODôoo was between 0.45 and 0.55 absorbance units. After log phase growth was reached the bacterial culture was transferred into sterile pre-cooled 50ml centrifuge tubes and cooled on ice for 30 minutes. The bacterial cells were then pelleted by centrifugation at 4000g for 15 minutes at 4°C. The supernatant was discarded and each pellet was resuspended in sterile-filtered, ice-cold buffer R Fl (lOOmM RbCl, 50mM MnCl], 30mM potassium acetate, lOmM CaCL and 15% (w/v) glycerol) (all reagents from Sigma, Poole, UK). For every 50ml o f bacterial culture, 5ml RFl resuspension buffer was used. The cell suspensions were incubated on ice for 20-25 minutes before re-pelleting the bacterial cells (2 500g, 15 minutes, 4°C). The Top 10

culture, 3.5ml of buffer RF2 was used. The resuspended cells were incubated on ice for a further 15 minutes before pipetting into 200-400pl aliquots in pre-cooled eppendorf tubes, then rapidly cooled to -80°C and stored at this temperature until required for use.

2.1.7 Transformation of plasmid DNA in to competent “Top 10” E. coli

A lOOpl aliquot o f competent Top 10 cells was incubated with a 1-lOpl volume of DNA (1 pi o f purified plasmid DNA or lOpl o f a ligation reaction) on ice for 30 minutes. The mixture was then heat-shocked for 90 seconds at 42°C, and then transferred back on to ice for 2 minutes. Subsequently, an SOOpl volume o f LB broth