Materials and Methods

2.1 Materials and Methods

2.1.1 Standard media and buffers

Unless o th e r wise s t a t e d solutions were sterilised by a u t o c l a v i n g a t ISpsi, 121 C for 20-25 min.

LB Medium (per 100ml)

Bacto-tryptone 1g, Bacto-yeast extract O.Sg, NaCI 1g, Glucose 0.1 g LB Agar

Bacto-tryptone 1g, Bacto-yeast extract O.Sg, NaCI 1g, I.Sg Bactor- ag a r

A n t i b i o t i c s

kanamycin sulphate was prepared a t a concentration of 2 5 mg/ml in distilled wate r and sterilised by filtration (0.22pim pore size). The working concentration was 2 5 ^ g /m l.

5XTBE 0.5M Boric acid, 0.5M Tris, lOmM EDTA 1XTE lOmM Tris, ImM EDTA pH 8.0

1XTNE lOOmM NaCI, lOmM Tris, ImM EDTA pH 8.0 20XSSC 3M NaCI, 0.3M Trisodium citrate

Gel loading buffer

0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol 45

Church hybridisation solution

0.5M NaPi pH 7.2, 7% SDS, ImM EDTA 1 M Sodium Phosphate solution pH7.2

Prepared by titration of 1 molar solutions of NagHPOA .^ZHzO and

NaH

2

0 4 2

20

Dénaturation solution 0.5M NaOH, 1.5M NaCI Neutralising solution

1.5M NaCI, IMTris, ImM EDTA pH 7.5

2 .1 .2 Suppliers o f Materials

R estrictio n e n d o n u c le a s e s

Restriction endonuclease were obtained from NBL or BRL

DNA modifying en zym es

Calf intestinal Boehringer Mannheim ph o sp h a ta se

T4 DNA ligase Anglian

Klenow DNA

polymerase New England Biolabs

Taq polymerase and buffer was supplied by Promega or NBL. Nucleotides were supplied by Boehringer Mannheim. All PCR was carried out in a Hybaid thermal reactor.

K i t s

Random prime

labelling kit Amersham International

Chemicals, reagen ts and membrane

Hybond N Amersham

[alpha-32-P] dCTP Amersham X-Ray film Kodak X-omat AR

Agarose Sigma

Bacterial media Difco

General Chemicals BDH and reagents

Cell lines

The hypoxanthine phosphoribosyl tr ansf er as e (HPRT) positive cell line, 6 4 0 6 3 a 1 2 a hamster-human hybrid containing a single copy of chromosome arm 9q (Jones and Kao, 1984) was provided by Carol Jones. The HPRT- hamster cell line, Wg3-h, (Goss and Harris, 19 75) was provided by Sue Povey. DNA from the DORA cell line which retains the Philadelphia chromosome was provided by Ben C a r r i t t

2 .2 General Techniques

2.2.1 Growth and expansion of cell lines

The cell lines 6 4 0 6 3 a 1 2 , Wg3-h and the radiation reduced hybrid lines were grown as at tached monolayers in Eagle's medium This was su pple m en ted with 10% v / v Foetal calf serum (PCS), 0.002M glutamine, non-essential amino acids, 0.1 M hepes, 0.2 %w/v NaHCOs and 100 units of Penicillin and Streptomycin in suitably sized flasks. The medium was brought to a slightly alkaline pH by the addition of a solution of NaOH/NaHCOg. Radiation reduced lines were also grown in HMT selection medium, lOO^m hypoxanthine,

10^im methotrexate and 10^m thymidine.

When th e cells were confluent they were washed in HANKS and released from th e flask surface by a brief incubation with a small volume of 0.25% trypsin. The trypsin was neutralised by t h e addition of so me fresh media and t h e cells were divided or transferred to a larger flask as required. Cells were stored in 1 ml aliquots in liquid nitrogen in 95% v/v PCS and 5% v / v DMSO.

Cell lines were subcloned a f t e r re su sc ita tion from liquid nitrogen. Cells were plated a t low density and colonies picked which were derived from a single cell. These colonies were ex pa nde d for DNA extraction. Cells were also retained for th e g e n e r a t i o n of m e t a p h a s e chrom oso m e s p r e a d s t o allow f o r fluorescent in situ analysis to t e s t for th e absence or retention of individual human fra g m e n ts p r e s e n t in th e p a r e n t irradiation hybrid line.

2.2.2 Isolation of DNA

2.2.2.1 DNA Preparation from Cell Lines.

Cells from 3 or more large flasks ( IS O c m ^ ) were thoroughly washed in HANKS. 7ml of lysing solution ( 0.5% SDS, lOOg/ml proteinase K in STB) was added to each flask and incubated for a t least 5 hours at 37°C or left incubating overnight a t 37°C. Protein

d e b r i s w a s r e m o v e d by t h r e e e x t r a c t i o n w i t h phenol:CHCl3 :lsoamylalcohol (2 5 :2 4 :1 ) followed by a further 2 e x t r a c t i o n s with CHCI3 to remove any tr a c e s of phenol. The nucleic acid was precipitated with 2 volumes of ethanol and t h e spooled DNA was air dried and resuspended in TE.

2.2.2.2 Large scale preparation of plasmid and cosmid DNA

400ml of LB broth with th e appropriate antibiotic selection was innoculated with a single bacterial clone and grown overnight with aeration in a 1 litre flask and harvested the following day by c e n tr ifu g a tio n a t 1 0 , 0 0 0 rpm for 10 minutes a t 4°C. The su p e rn a t a n t was discarded and the pellets resuspended in a total volume of 30ml Solution 1 (SOmM glucose, lOmM EDTA, 2 5mM Tris.CI (Ph 8.0), 4mg/ml lysozyme) and allowed to sta n d a t room te m p e ra tu re for 5 minutes. 60ml of freshly prepared solution 2 (0.2 M NaOH, 1% SOS) was added and the mixture was gently mixed prior to incubation on ice for 5 minutes. 45ml of ice-cold solution 3 (3M potassium a c e ta te ) was lastly added followed by a further incubation on ice for 5 minutes. After lysis, the cell debris was pelleted by centrifugation a t 1 0 , 0 0 0 rpm for 15 minutes and th e su p e rn a ta n t retained.

0.6 volumes of propan-2-ol, approximately 70ml, was added t o precipate th e DNA. After a 4 0 min period of incubation t h e pr ecipitat e was pelleted a t 1 0 0 0 0 rpm for 15 minutes a t room temperature, drained and resuspended in 11.2 ml of TE buffer. To this suspension was added 0.4 ml of EDTA, 0.4 ml Tris Base, 12g of solid cesium chloride (CsCI) and 1.2 ml of 10mg ml ethidium bromide. The solution was thoroughly mixed and spun a t 5 0 0 0 rpm for 5 minutes to remove any remaining precipitate and pip etted into Sorvall polyallomer ultracentrifuge tu be s. The t u b e was completely filled with parrafin oil and the tube was sealed with a Sorvall ultracrimp tub e sealer. The tu be s were spun in a Sorvall u ltr ac e n tr if u g e a t 4 5 0 0 0 rpm a t 20°C for 18 hours. A f t e r centrifugation the plasmid DNA was visualised under UV light and removed using a syringe. The ethidium bromide was removed from

th e plasmid DNA by r e p e a te d extraction with C s C I - s a t u r a t e d propan-2-ol and th e CsCI was then removed by dialysis overnight in 5 litres of TE buffer.

The DNA was precipitated (section 2.2.1.3) and recovered by centrifugation. The pellet was washed in 70% ethanol, dried and resuspended in a suitable volume of TE. The concentration of the DNA was determined by s p e c tro p h o to m e try a t 2 6 0 n m ( s e c t i o n 2.2.1.4.).

2.2.3 Preparation and transformation of co m p ete n t E.coli

C o m p e t e n t E.coli JM101 cells, were p r ep ar ed using t h e Calcium chloride (CaCle) procedure as described by Maniatis e t al. (1 9 8 2 ). JM101 were grown on a miminal media plate to preserve th e F’ episome containing the ge nes for proline synth esi s (this includes t h e g e n e requir ed for b l u e / w h i t e s e l e c t i o n of recombinants). 30ml of L-broth was innoculated with 0.5 ml of a fresh overnight culture of JM101 and incubated a t 37°C with shaking until the culture reached an OD 550 of 0.4-0.6. The culture was chilled on ice for 10 min and pelleted by centrifugation a t 5 0 0rpm a t 4°C. The cell pellet was re-suspended in 1 5ml of ice- cold, sterile, 50mM CaCI^ solution and kept on ice for 15 min. Cells were pelleted as before and resuspended in 3ml of ice-cold, sterile, 50mM CaClg^. The re-suspended cells were stored on ice before being transformed.

For transformation, generally up to 20^1 of DNA solution, a p p r o x i m a t e l y 50-1 OOng, was ad de d to a 200^1 aliquot of co m p e te n t cells, incubated on ice for 40 min, heat shocked a t 42°C for 9 0 seconds and reincubated for a further 2 min on ice, before plating o u t on L-agar containing t h e a p p r o p r i a t e antibiotic resistance. When using Amp resistance vector syst em s, th e cells were incubated in 0.5 ml of L-broth for 30 min prior to plating to allow for pre-expression of th e Amp resistance. When using blu e /w hit e selection screening, 50^1 of 2% X-Galactosidase and 20jil of lOOmM Isopropylthiol-beta-D-galactoside was ad d e d to

t h e t r a n s f o r m e d cells be fore plating. The t r a n s f o r m a t i o n efficiency of th e cells was t e s t e d by transforming with lOng o f undigested vector DNA. The efficiency of the CIP reaction (section 2 . 2 . 2 . 2 . ) for p r e v e n t in g self religation was e x a m i n e d by transforming with dephosphorylated vector.

2 .3 DNA Analysis

2.3.1 Agarose gel electrophoresis

DNA digestion products were resolved in 0 .4 - 2 .0 % a g a ro s e gels prepared and run in IxTBE buffer. Samples were loaded with the addition of 0.1 vol of lOx loading buffer. The DNA was stained with ethidium bromide a t 0.5 ^g/ml and visualised under ultra­ violet transillumination.

2.3.2 Precipitation of DNA

DNA was precipitated by the addition of 0.1 x volume of 3M NaAc followed by 2 x volume of ice-cold ethanol. The DNA solution was in c u b ate d a t -70°C for 5 minutes t o allow t h e DNA t o precipitate. The DNA was pelleted by centrifugation a t 1 0 0 0 0 rpm or in a bench centrifuge as appropriate for 10 minutes. The su p e rn a tan t was discarded and the pellet washed in 70% ethanol before being dried and resuspended in an appropriate volume of TE, usually

2.3.3 Recovery of DNA fragment from agarose gels

DNA fra gm ents were isolated from agarose gels using t h e Gene Clean method. The DNA fragment of interest was e x c i s e d from the gel and the agarose melted by incubation in 3 x vol Nal and 1 X TBE modifier at 50°C for 5 min. Glassmilk was added a n d the suspension was incubated on ice for 5 minutes to allow binding of th e DNA and Glassmilk together. The glassmilk-DNA complex

was pelleted by centrifugation for 5 seconds and washed in New Buffer X 3 before the DNA was eluted in double distilled w a t e r or TE.

2.3.4 Concentration of DNA

The c o n c e n t r a t i o n of t h e DNA w a s m e a s u r e d by s pe ctropho tometry in a CIBA CORNING 2 8 0 0 Spectrascan a t 260nm or es tim a te d by direct comparison of a small am oun t of th e DNA with a known amount of lamda ladder after gel electrophoresis.

2 .4 DNA Modification Reactions

2.4.1 Restriction digestion of DNA

Restriction enzyme digestion of plasmid and cosmid DNA was generally carried out in a reaction volume of 20^,1 using O.S^g of DNA. Reaction buffer and restriction enzymes were th o se supplied by th e manufacturer and incubation was typically for a period of 2 hours under the specified conditions. For the digestion of genomic and hybrid DNA reactions similar to t h a t described above were s e t up but the reaction volume was increased to a total volume of 40- 60^1 and incubation continued overnight.

2.4.2 Dephosphorylation of DNA

The terminal phosphate group of linearised vecto r DNA was removed, using calf intestinal phosphatase (CIP), to pr ev en t self ligation of th e ends in subsequent cloning experiments. In general, 2- 5ng of vector DNA was digested with th e appropriate restriction e nz ym e in One-Phor-AII Plus buffer. 0.1 units of CIP was a d d e d and incubated a t 37°C for 30 min and the reaction stopped by hea t inactivation of th e enz ym e at 85°C for 15 min. The CIP was removed by 2 phenol followed by 1 chloroform extraction and the DNA was recovered by ethanol precipation, th e pellet washed in 70% ethanol, dried and resuspended in a suitable volume of TE.

2.4.3 Ligation of DNA

Insert DNA was ligated t o t h e a p p r o p r i a t e c u t an d pho sp ha tase d vector in a solution containing 1 OOng of vector was used for most subcloning operations. Reactions were performed at 1 4°C overnight and generally contained a t least a 3-fold molar excess of insert DNA to vector DNA with one unit of T4 DNA ligase.

2 .5 Radioactive Labelling of DNA probes

A commercial labelling kit was used (Amersham). Usually 2 0 - 5 0 ng of insert DNA or Alu PCR products were labelled. The DNA was denatured by incubation a t 99°C for 5 minutes in a Hybaid PCR a u t o m a t e d heating block. Following a brief incubation on ice, lOul of buffer 1 ( dATP, dGTP, dTTP and labelling buffer) and 5ml of buffer 2 (containing random hex anucleotide primers) were added. 50 Ci radiolabelled nucleotide alpha 3 2 p dCTP (lOCi/pl) was added to the reaction mix followed by 2.5^il (2.5 units) of DNA polymerase 1, klenow fragment to give a final volume of 50^1. Incubation was for a t least 3 hours a t room temp erature or a t 37°C for b e t w e e n 1-3 hours a f t e r which tim e t h e re a c tio n was terminated by the addition of 50ml of TNE/0.1% SDS.

Unincorporated nucleotides were removed using a Sephadex G50 column. Sephadex suspended in TNE/0.1% SDS was c o m p a c t e d by centrifugation a t 200 0 rp m for 2 minutes into a 1 ml syringe. The labelling reaction was layered onto this and the spin repeated. The labelled DNA was c ontain e d in t h e e l u a te . Prior t o hybridisation the probe was denatured by boiling generally in the presence of 1 mg of sonicated herring sperm DNA and cooled on ice.

2 .6 Southern analysis

A fte r elec tro p h o r esis in 0.8 % a g a r o s e gels, DNA w a s depurinated by submerging the gel in 0.25M HCL for 20 min. The gel was rinsed in distilled water and soaked in denaturing solution (1.5M NaCI, 0.5M NaOH) for 2 x 20 min followed by a further 2 x 20 min in neutralising solution (1.5M NaCI, 0.5M Tris/HCL, pH 7 . 2 ) , prior to Southern blotting of the DNA on to nylon membrane, Hybond N, in 20 X SSC for 16 hours. After gentle washing in 2 x SSC, the

filters were baked a t 8 0 ° C for 2 hours. Filters were p re ­ hybridised and hybridised in Church prehybridisation solution (0.5M NaPi, 7%SDS and ImM EDTA). Pre-hybridisation was for >2 hours and hybridisation was for 14-16 hours following t h e addition of 3 2p-|abel led DNA probe. Restriction f ra g m e n t s for South ern analysis were 32p_|abelled by random priming (section 2 .2 .7 ) . Filters were washed in 2 x SSC, 0.1% SDS for 2 x 2 0 min a t 65°C and in 0.1 x SSC, 0.1% SDS for 30 min a t 65°C when appropriate, before autoradiography at -70 °C.

Before reuse filters were stripped of their radioactivity by incubation in 0.4M NaOH for 15 min followed by neutralisation in 0.2M NaPi pH 7.2, 0.1 x SSC and 0.1% SDS.

2 .7 Preparation o f c o s m id libraries from individual hybrid lines

2.7.1 DNA Preparation

DNA was prepared from the radiation reduced hybrid lines 178 and 20A which retain a small amount of human DNA from th e region surrounding t h e TSC1 locus. Hybrid DNA was partially digested with the restriction Mbol and size fractionated by sodium chloride density gradient. DNA from the fractions retaining 3 5 - 4 5 Kb f ra g m e n t sizes were ligated into the cosmid v e c to r Lorist B (Cross and Little 1986). Vector arms were prepared according t o the conditions described by Little (1987). Approximately 500ng of hybrid DNA was ligated to 200ng of vector in an reaction volume of lOul.

2.7.2 Packaging reaction

The lin e ar c o sm id m o le c u le s w e re p a c k a g e d in to b acteriophage lamda head particles in th e following reaction: 2ul of th e ligation reaction was mixed with 7ml of buffer A (20mM Tris.CI pH 8 .0 , ImM EDTA, 5mM MgCI, 0 .0 5 % v / v B- m e rc a p to e th an o l) and lul of buffer Q (6mM Tris.CI pH7.5, 18mM MgCI, 60mM spermidine pH7.5, ISmM ATP pH7.6, 0.2% v /v B- m ercaptoethanol). 4^1 of sonicated ex tract and 5^1 of freeze thaw lysate were added and incubated for 1 hour at 30°C. The reaction volume was made up to 2 0 0^il with phage storage medium and th e phage were used to infect th e recom binant deficient strain of