PRODUCCIÓN GANADERA a) Cordero

Two bacteriophage libraries were used in this study: the LS normal colonic mucosal library (LSNCM) described above and a chromosome 5 specific genomic library. The chromosome 5 library (LAO5NS01) was purchased from the American Type Culture Collection (ATCC). This library was prepared from the complete EcoRI digestion of a human/hamster hybrid cell line (640-12), cloned into Charon 21A, with an average insert size of 4k b.

2.51. Plating a Bacteriophage Library onto L-Agar.

The cDNA library LSNCM was plated onto E.coli. NM514 cells prepared as before, whilst the genomic library was plated onto E.coli. LE392, which were prepared as follows:

10ml L-broth (appendix 1 .2a), 0.2% maltose (w:v)’ was inoculated with E.coli. LE392 and grown overnight at 37°C,

the cells were harvested and resuspended in 4ml cold lOmM MgS0 4.

For each library two 24.5cm square tissue culture tray dishes (Nunc. BRL) containing L-agar (appendix 1.2a) were used, =100, 000 plaques could be plated onto each plate. An appropriate dilution of the phage suspension was incubated with SOOfll plating cells and incubated at 37°C for 15 minutes, this was then mixed with 50ml molten top-agar

(appendix 1.2a) , and poured onto the plate, it was then inverted and incubated overnight at 3 7°C.

2.52. Transfer of Plaques to Nylon Hybridization Membrane. The plates were cooled at 4°C for at least an hour prior to transfer to the nylon membranes (Hybond-N, Amersham International Pic). A piece of Hybond-N was placed carefully on top of the agar for 1 minute, during which time the membrane was pierced with a hypodermic needle for subsequent realignment, the Hybond-n was removed and placed plaque side up onto a piece of Whatman 3mm paper soaked in 1 . 5M N a C l , 0 . 5M NaOH. The plaque dénaturation was allowed to proceed for 3 minutes, when the membrane was neutralized for 7 minutes by transfer to a piece of 3mm paper soaked in 0 . 5M Tris.HCl pH 7.2, 1 . 5M NaCl, ImM EDTA. The membrane was then rinsed in 2xSSPE (appendix 1.3) and allowed to air dry. Finally the membrane was baked at 80°C for 2 hours and the DNA was cross linked by ultraviolet light for 3 minutes on a transilluminator. A second replica membrane of the plate was routinely taken as above, except that it was left in contact with the plaques for 90 seconds and the alignment marks of

the first membrane were copied onto the second.

2.53. Prehybridization, Hybridization and Posthybridization Treatment of Hybond-n Plaque Replicas.

The membranes with plaques immobilised on them were processed as described in appendix 2,4b.

The chromosome 5 specific genomic library was probed with an aliquot of the cDNA library LSNCM which had been radiolabelled using the polymerase chain reaction (PCR). The reaction was as described in section 2.55c, except that l}ll of the amplified library (5x1 Q H pfu/ml) was the template and 50|J,Ci 5 ' [a-32p]dCTP were added to the reaction during the

elongation phase of the twelfth cycle. After a total of 30 cycles the reaction was extracted with chloroform and applied to a G50 Sephadex spin column as described in appendix 2.4d.

2.54. Second and Third Round Screening.

After the first round of library screening had been performed, successive rounds were required in order to identify single positively hybridizing plaques. Thus, a hybridizing region present on the first round of screening, was physically removed as a 1cm diameter plug of agar from the bioassay tray. This plug was placed into 1ml SM (appendix 1.2a) , to which a drop of chloroform had been added and the phage particles were allowed to diffuse from the agar for at least an hour at 4°C. The phage suspension was titrated to result in a dilution which gave approximately 3 00 plaques per 90mm petri dish. Sections 5.52 & 3 were then repeated and any positively hybridizing plaques were removed as plugs with the tip of a glass Pasteur pipette. These plugs were again suspended in 1ml SM buffer, plus chloroform, and the whole procedure was repeated once more, until individually hybridizing plaques could be reliably identified.

2.55. Analysis of Plaque Purified Bacteriophage Clones. 2.55a. Small Scale Isolation of Bacteriophage DNA.

Small scale DNA preparations were performed on phage suspensions prepared from plate lysates. A 90mm petri dish was plated with a dilution of a single plaque suspension to give a confluent lawn of plaques on the relevant plating cells. After incubation overnight at 37°C, the plate was overlayed with 4ml SM buffer as described in section 2.43 above. DNA was isolated from the resulting phage suspension precisely as outlined in the XgtlO cloning system booklet

(Amersham International pic), using polyethyleneglycol-6000 (PEG)/NaCl precipitation and DEAE cellulose DE52 (Whatman). The final product of between 20 and 50|lg was resuspended in

50|ll TE pH 7.4 buffer.

2.55b. Medium Scale Isolation of Bacteriophage DNA.

A medium scale preparation was used to obtain a higher yield of a purer product. A 24.5cm tissue culture tray was plated to confluence with a dilution of a single plaque

suspension. After incubation overnight this was overlayed with 3 0ml SM buffer to obtain a phage suspension as above. Chloroform lysed bacterial debris was removed from the suspension by centrifugation at 4000g for 10 minutes at 4°C. The phage particles were precipitated from the supernatant by the addition of 2.8g PEG6000 and 1.2g NaCl, and incubation at 4°C for at least 6 hours. The phage were then sedimented at 3000g for 10 minutes and the resulting pellet was resuspended in 10ml TM (lOOmM Tris.HCl pH 7.4, 50mM MgCl2 ) . 7 . 5g CsCl were added and the s u spension was s u b j e c t e d to ultracentrifugation at 35000rpm for 16 hours at 15°C. This resulted in opalescent band of phage particles, which was removed by puncturing the side of the centrifuge tube with a 21 gauge hypodermic needle attached to a syringe. Ultracentrifugation was repeated, following dilution of the phage suspension with TM, CsCl (0.75gml'l). The opalescent band was again removed and transferred to dialysis tubing,

for dialysis against lOmM Tris p H 7 .4, lOmM MgCl2 overnight at 4°C. The phage particles were lysed by the addition of EDTA to lOmM and SDS to 0.1%(w:v) and the DNA was extracted twice with phenol and once with chloroform. Finally the DNA was removed as a precipitate following the addition of 2.5 volumes ice cold ethanol.

2.55c. Polymerase Chain Reaction Amplification of X gtlO Insert.

DNA cloned into the EcoRI site of ÀgtlO could be amplified directly from phage suspensions, with primers flanking the cloning site as shown below. Thus providing a pure source of insert DNA.

Forward: 5'-TGAGCAAGTTCAGCCTGGTTAAGTC-3' Reverse : 5'-GGTGGCTTATGAGTATTTCTTCCAG-3'

A single plaque was picked from an agar plate into 1ml SM with the tip of a glass Pasteur pipette. A drop of chloroform was added and the phage were allowed to diffuse

for at least an hour at room temperature. l|ll of the suspension was added to 26|ll H2O in a 0.5ml microcentrifuge tube and frozen at -70°C for 10 minutes, when the tube was plunged into a boiling water bath and boiled for 3 minutes.

The standard PCR mix (appendix 2.7) and 50pM of each of the above primers were added to the still hot phage suspension to bring the total volume of the reaction up to 100|Lll. The DNA was amplified after a 5 minute incubation at 95°C and addition of 1 unit Tag DNA polymerase (Promega) through 30 cycles as follows: 90°C for 30 seconds, 50°C for 30 seconds and 7 0°C for 2 minutes. The products of the reaction were analysed on 1.2% agarose (w:v) TAE gel (appendix 2.2a).

2.55d. Subcloning Bacteriophage Derived Inserts into Plasmid Vectors.

For ease of handling, inserts derived from phage clones were routinely subcloned into one of the pUC series of plasmid vectors (Vieira & Messing, 1982, Yanish-Perron, et al, 1985) . Five nanogrammes of EcoRI digested pUC vector DNA were ligated to approximately l[lg EcoRI digested phage DNA in

the presence of 50mM Tris.HCl pH 7.8, lOmM MgCl2 , ImM ATP, 2OmM dithiothreitol, SOjlgml"^ bovine serum albumin with 1 unit T4 DNA ligase (Bdehringer Mannheim, Sussex) in a total volume of 10|Il. The reaction was allowed to proceed overnight at 11°C followed by a 30 minute incubation at 37°C and heat inactivation at 68°C for 10 minutes. The whole ligation reaction was added to the competent cells for transformation as described in section 2.31b.

Recombinant colonies were identified by plating the transformation mix onto L-agar plates which had been o v e r l a y e d w i t h 3 ml L - a g a r c o n t a i n i n g ; 2 0 )1 1 isopropyl thiogalactoside (IPTG)(1M), 40)11 5-bromo-4-chloro-3- indolyl-|3-glucopyranoside (X-gal ) ( 2Omg/ml ) and 3|ll ampicillin

(200mg/ml). Non-recombinants appeared as blue colonies, whilst recombinants were white. Small scale plasmid DNA preparations (section 2.32) were performed on a number of white colonies to check that they contained the insert of interest, once this had been established a large scale plasmid DNA preparation was under taken.

2.6. Analysis of Clones Isolated From the Chromosome