Percepción grupal de pertenencia a una red de “Comunidades Íñigo”

After growth of the DH5a cells containing the rat brain cDNA library the plates were removed from the incubator and labelled as the master plates. Nylon membranes were cut to cover the size of the plates, labelled and marked in one comer for orientation with a soft lead pencil. Each membrane was dampened by placing it onto a freshly prepared LB ampicillin agar plate. The dampened membrane was placed apposed to the colonies on the master plate using tweezers and 1 min allowed for colony transfer. The position o f the

membrane was outlined using marker pen on the base of the original master plate to aid realignment when selecting colonies containing positively hybridizing clones. The membrane was carefully lifted again using tweezers and placed colony side-up on a new LB ampicillin agar plate. Both the master and replica plates were incubated at 37°C for 18 h for colony growth. Subsequent rounds of screening used smaller LB ampicillin agar plates (90 mm^) where the library was plated directly onto circular nylon membranes cut to size and the replica plates were produced by membrane to membrane transfer of the colonies. The circular membranes were trimmed on one side to provide a straight edge which would aid orientation when selecting positive hybrid colonies.

2.3.53 ALKALINE BACTERIAL CELL LYSIS ON NYLON MEMBRANES After replica plating, the original master plates were refiigerated for subsequent use in colony selection. The replica plates were treated in the following manner to achieve bacterial cell lysis. Sufficient pieces o f blotting paper (3MM) were cut to fit 6 bioassay plates (24 cm^). A stock solution o f 20 x standard saline citrate (SSC; 3 M NaCl, 300 mM sodium citrate, pH 7.0) was prepared and diluted as necessary for all washing steps. Each individual piece of blotting paper was soaked in one of the following six solutions :

1) 10% (w/v) SDS

2) 1.5 M NaCl, 0.5 M NaOH

3) 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl 4) 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl

5) 2 X SSC (i.e., 300 mM NaCl, 30 mM sodium citrate, pH 7.0 ), 0.5% (w/v) SDS 6) 2 X SSC

All excess solution was removed and each of the six plates labelled to denote the solution in which the blotting paper had been soaked. The nylon membranes were exposed to solutions 1- 6 consecutively, by being placed on each piece of blotting paper colony side up to avoid any disruption o f the colony positions. They were incubated for 4 min in solution 1 (10% SDS) and 5 min in all the remaining solutions (2-6). The treated nylon membranes were then placed onto a dry piece of 3 MM blotting paper colony side up to remove excess fluid and covered with plastic film. DNA from the lysed bacterial cells was cross linked to the nylon membranes by exposing them to UV radiation at 1.2 joules/cm^

2.3.5.4 PREPARATION OF A [a^^P] dCTP RADIOLABELLED cDNA PROBE Primer labelling mix (1.25 M Tris-HCl, pH 8.0, 125 mM MgCl2, 250 mM p~ mercaptoethanol, 500 mM dATP, 500 mM dTTP, 500 mM dGTP, 1 M HEPES buffer, pH 6 .6, 4.5 pg random primers: 5 pi), was added to the purified DNA (50-70 ng) to be amplified and made up to 25 pi in H2O. The DNA was denatured by boiling the entire sample in a water bath. The reaction mixture was chilled on ice for 1 min and 2 pi of Klenow polymerase (2 U/pl) added. Subsequently, 4 pi of [a^^P] dCTP (10 mci/pl) was added and the mix was incubated for 2 h at 37°C or alternatively at room temperature overnight. After incubation, the labelled probe was precipitated by addition o f 15 pi o f H2O, 200 mM EDTA (10 pi), 30 mg tRNA, 1.6 M NaCl ( 8 pi) and 100 % ethanol (154 pi). The tubes contents were mixed and then incubated on dry ice for 15 min. After incubation the tube was centrifuged at 10000 x g for 10 min. The supernatant was decanted and the DNA pellet resuspended in 112 pi o f sterile water. An aliquot (2 pi) o f the [a^^P] dCTP was diluted in 1 ml of scintillant and the radioactivity measured in the scintillation counter. The specific activity of the probe was calculated. A final probe concentration o f 2 x 1 O^cpm/ml was recommended for northern and Southern blotting and 5 x lO^cpm/ml for colony hybridisations. The specific activity of the probe was calculated as (cpm/pg)cpm x dilution factor X 55pl/ pg o f DNA used in labelling reaction The radiolabelled probe was used immediately or stored at -20°C overnight. It was denatured for 5 min at 100°C prior to use and chilled rapidly on ice. Hybridisation temperatures for the radiolabelled probe were calculated by the equation: Td= 81.5°C + 16.6 (loglO[Na+]) + 0.41 [(G+C)(100)]/n -500/n, where n represented the number of nucleotides (Sambrook et al., 1989)

2.3.5.5 HYBRIDISATION OF A [a^^P] RADIOLABELLED cDNA PROBE TO NYLON MEMBRANES

After DNA released by the alkaline lysis o f bacterial colonies was cross-linked to nylon membranes (2.3.5.3), residual bacterial cell debris was removed by three consecutive washing steps. Membranes were transferred to a polypropylene container and agitated at 70 rpm with 6 x SSC (200 ml) at 37°C for 5 min. The solution was substituted with 6 x SSC/SDS ( 6 X SSC, 0.5 % (w/v) SDS: 200 ml) and the membranes agitated at 37°C for 5 min. The membranes were incubated for a further 20 min in 200 ml o f fresh 6 x SSC/SDS

at the calculated hybridisation temperature (2.3.5.4) with shaking at 70 rpm. The final wash solution was removed and the membranes covered with prehybridisation solution ( 6 x SSC, 0.5% (v/v) SDS, 400 fig of herring sperm DNA, 0.1% (w/v) ficoll, 0.1% (w/v) polyvinyl- propylene, 0.1 % (w/v) bovine serum albumin: 100 ml) and incubated at 65°C for 4-5 h. The prehybridisation fluid was replaced and included the denatured [a^^P] labelled cDNA probe. The nylon membranes and fluid were covered with plastic film to prevent evaporation of the solution and incubated overnight at the calculated hybridisation temperature, with shaking at 70 rpm in a hybridisation oven.

After hybridisation, the membranes were removed from the oven and washed twice with 100 ml of 2 X SSC/ 0.1% (w/v) SDS at room temperature and then 100 ml of 0.2 x SSC/ 0.1 % (w/v) SDS at 65®c for 15 min with shaking at 70 ipm. The membranes were placed on 3 MM blotting paper to remove any excess fluid for 15 min. They were then transferred to dry 3 MM blotting paper, covered with plastic wrap and placed against a phosphorimaging screen which detected radioactive beta emission. The emissions were later visualised using a Molecular Dynamics Phosphorimager in conjunction with the Image Quant software package.

2.3.6 NORTHERN BLOTTING

A commercial rat multiple tissue northern blot (Clontech, Hampshire UK) was used for northern blot analysis where hybridisations were conducted according to the manufacturers recommended protocol. Each lane of the northern blot contained 2 pg o f poly A+ RNA from the following rat tissue sources respectively: heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis.

2.3.6.1 HYBRIDISATION OF A [a^^P] RADIOLABELLED cDNA PROBE TO A MULTIPLE TISSUE NORTHERN BLOT

The solution used for hybridisation was ExpressHyb™ (Clontech, Hampshire UK). At room temperature the solution appeared turbid and therefore was warmed to 6 8®C to dissolve any precipitate. The northern blot membrane was incubated at 6 8®C with agitation in ExpressHyb solution (5 ml) for 30 min. A [a^^P] radiolabelled cDNA probe was prepared as described in (2.3.5.4) and added to ExpressHyb (5 ml) and incubated with the blot for 1 h at the calculated hybridisation temperature. The blot was washed repeatedly in 2 x SSC/

0.05 % SDS for 40 min and then incubated at 50°C with continuous agitation in 0.1 x SSC/ 0.1 % SDS. After 40 min, the northern blot membrane was removed from the wash solution using forceps and shaken to remove excess fluid. The membrane was covered in plastic wrap and mounted on 3 MM blotting paper and then placed against a phosphorimager screen overnight. Any resulting beta emission from the radiolabelled blot was visualised using the Molecular Dynamics Phosphorimager in conjunction with Image Quant software.

2.3.7. SOUTHERN BLOTTING

Flat-bed agarose gel electrophoresis of DNA samples was undertaken as described in 2.3.2.1. The agarose gel was then carefully removed from the electrophoresis chamber and immersed in 0.25 M HCl for 15 min. The HCl was removed by rinsing with dH2 0 and was replaced with a second alkaline solution (1.5 M NaCl, 0.5 M NaOH) in which the gel was immersed for 30 min with agitation. The second solution was removed by rinsing the gel in dH2 0 again and the gel was immersed in 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.2, 1 mM EDTA for 30 min. A plastic tray was filled with 20 x SSC solution (2.3.5.3) into which a raised platform was placed. The platform was covered with three pieces of 3 MM blotting paper which would draw up the 20 x SSC solution in which they were partially immersed, by capillary action. The gel was placed onto the platform and covered with nylon membrane which was sized exactly to the gel. Care was taken to avoid the trapping o f air bubbles between the gel and nylon membrane which could interfere with DNA sample transfer. Three pieces o f 3 MM blotting paper were also sized to the gel and placed on top of the nylon membrane. A stack of absorbent towels was placed over the blotting paper and the stack held together with a weighted (0.75-1 kg) glass plate. The 20 x SSC solution was covered with plastic wrap to prevent evaporation. The transfer was allowed to proceed overnight at room temperature. The nylon membrane was removed and rinsed in 2 x SSC to remove any adhering agarose. Detection of the DNA was by hybridisation of a radiolabelled cDNA probe as described in (2.3.6.1).

2.3.8 DATABASE SEARCHES AND SEQUENCE ANALYSIS

2.3.8.1 SEQUENCE ALIGNMENT TOOLS

Nucleotide and amino acid sequence similarity searches were conducted using the basic local alignment search tool, BLAST 2.0, (Altshul et al, 1990). BLAST 2.0 constitutes an

algorithm for sequence comparison allowing the rapid search of databases for alignments to a submitted query. Nucleotide sequences were submitted in text format and used blastn, (a nucleotide-specific search) to search the GenBank, expressed sequence tag (EST) database, the DNA database of Japan (DDJB), and European Molecular Biology Laboratories (EMBL) DNA database for nucleotide identity. Protein sequences were submitted in single letter amino acid code, also in text format and used blastp (a protein- specific search) to search S WISSPROT and PDB databases for protein sequence alignment. BLAST 2.0 was accessed via the National Centre for Biotechnology Information (NCBI) website at http://www.ncbi.nlm.nih.gov/BLAST.

2.3.S.2 DETERMINATION OF INTRON-EXON COMPOSITION OF GENES