2 1 4 NORTHERN ANALYSES

A 1.5 % agarose gel was prepared (1 4 x 1 1 x 0.7 cm), in 20 mM MOPS (3-[N - m orpholinoj-propane-sulfonic acid. Sigma), 5 mM NaAc 3HzO, 1 mM EDTA (pH 7), 18% form aldehyde (BDH). 10 or 20 /xg samples of total cellular RNA or A+ RNA prepared from 33.3 /xg cellular RNA were heated at 65 “C in 14 /xl n o rthern dye mix (10 % 10 X northern running b u ffer (table 2.2), 62.5% deionised formam ide, 18% form aldehyde.

0.075% xylene cyanol, 0.025% bromophenol blue, 5% glycerol, 0.1 mM EDTA). A fter 5 min, the samples were quenched on ice, 0.5 /xg of ethidium bromide added, and samples loaded alongside 5 /xg of 0.24-9.5 Kb RNA ladder (BRL). The gel was electrophoresed in 1 X n orthern running buffer, 10 % formaldehyde at 30V overnight.

The gel was photographed on Polaroid 667 film while illuminated on a TM20 UVP 220 uv transillum inator. Prior to dénaturation of the RNA in 50 mM NaOH, 10 mM NaCl, the gel was soaked in several changes of water. A fter 20 min of dénaturation, the gel was rinsed in H^O and neutralised for 45 min in 500 mM Tris-H C l (pH 7), 1.5 M NaCl.

T ransfer of the RNA to nylon membranes (Amersham Hybond N) was achieved by

capillary blotting for 12 hours. The gel was lain on top of a 3mm Whatman filter soaked in 10 X SSC, with wicks dipping into a tank of 10 X SSC. The gel was overlain with Hybond N and 2 pieces o f 3mm also soaked in 10 X SSC, followed by a stack of dry paper towels, a glass plate and a 500g weight. Evaporation was reduced by covering the tank with

Saranwrap.

Efficiency of transfer was checked by uv illumination of the ethidium bromide stained samples on the Hybond N, and the position of migration of the RNA ladder and ribosomal bands was marked on the filter. The filter was uv crosslinked for a total of 3 min and baked at 80®C for 2 hours.

Pre-hybridisation was performed in 10 ml o f hybridisation buffer (table 2.2 ) in heat sealed platic bags in a thermostatically controlled shaking H^O bath at 65°C. A fter 1 hour, 2.5 X lO^cpm/ml hybridisation buffer of ^^P labelled riboprobe, 200 /xg/ml sonicated salmon sperm and 100 /xg/ml yeast tRNA were heated to 95®C for 5 min and quenched on ice before addition to the hybridisation mixture. Hybridisation was perform ed for 12 hours at 65°C. Filters were washed in 1 X SSC, 1% SDS for 30 min at 65®C followed by 30 min in 0.1 X SSC, 1% SDS at 65°C, followed by an additional 15 min at 70°C for the p46 RNA tissue distribution rt-prthern (Chapter 7, figure 7.2C).

Damp filters were autoradiographed for 96 hours (p46 360), and 48 hours (human 7 actin), and 72 hours for the A+ northern.

2.15. RNAse PROTECTION ANALYSES

20 /xg RNA samples were mixed with human 7 actin and p46 360 gel purified riboprobes in 40mM Pipes (pH 6.4, BDH), 1 mM EDTA (pH 8), 400 mM NaCl, 54 % deionised formam ide. Samples were denatured for 5 min at 90®C, and annealed for 12 hours at 55®C. Samples were treated with 8 /xg/ml RNAse A (Boehringer M annheim), and 40 U /m l RNAse T1 (Boehringer Mannheim) in 300 mM NaCl, 10 mM Tris-H C l, 5 mM EDTA (pH 8) for 1 hour at 37°C. RNAse was destroyed by addition of 5 /xg proteinase K in 0.6% SDS for 15 min at 37®C, and RNA was phenol chloroform extracted and

precipitated with 20 /xg yeast tRNA and ethanol on dry ice for 20 min. RNA was collected by centrifugation (20 min at 13 K rpm in a Sartorius microfuge), and dried under vacuum

for 3 min before addition of 5 /xl of 80% deionised form am ide, 5% glycerol, 0.1 mM EDTA, 0.025% bromophenol blue and 0.075% xylene cyanol. Samples were loaded onto a pre-ru n 4% polyacrylamide, 7M urea, IX TBE sequencing gel, and electrophoresed in 1 X TBE at 40 W for 2 hours with labelled pBLcû/2 Hinfl^ EcoRl as markers. The gel was dried onto 3mm Whatman paper, and autoradiographed for 1 hour ( 7 actin signal) and 12 hours (p46 360 signal).

2.16. IN SITU HYBRIDISATION

The protocol for the preparation and sectioning of mouse embryos (CBA/Ca) and the in situ hybridisation details were taken from Wilkinson and G reen (1990). Probes used were [UTP] labelled sense and antisense p46 360 riboprobes (section 2.9.5),

hybridisation was performed at 55”C, and washes were perform ed under high stringency conditions at 65®C. Ilford K5 emulsion was used to dip the slides for autoradiography under safelight conditions (sepia filter no. 914 from Ilford), and slides were developed in K odak D19 developer after 17 days exposure at 4°C.

Sections were photographed using Kodak Ektachrome 5018 EPY 64T film , using a Zeiss photomicroscope III with illumination set at 12V. Bright field photographs were automatically exposed at ASA 25, and dark field photographs were taken after 2 sec exposure (6.5, 8.5 day embryos), 6 sec exposure (10.5 day embryos) and 8 sec exposure (12.5 and 14.5 day embryos) at ASA 100.

2.17. PROTEIN EXTRACTS

2.17.1. P reparation of Manley whole cell extracts

Whole cell extracts were prepared from 5mls of packed cells, exactly as described by Manley et al., 1980.

2.17.2. Preparation of large scale Manley extracts.

Large scale whole cell extracts for protein purification were prepared from 47 ml of packed cells, as above, except that solution C was replaced by 0.1 M col.buffer (table 2.4).

2.17.3. Preparation of Scholer micro whole cell extracts

Whole cell extracts from small numbers of cells or small quantities of tissue were prepared using the buffer described by Scholer et al., 1989, except that 0.5 m g/m l protease inhibitor (Sigma), and 1 mg/ml trypsin inhibitor (Sigma) were used in place of pepstatin as protease inhibitors.

either used immediately in gel retardation assays, or were frozen on dry ice, and stored at -70®C.

2.17.4. Preparation of micro nuclear extracts

Small nuclear extracts of tissues and cells were prepared by resuspending material in 400/il of b u ffer A described by Schreiber et al., 1989, with added protease inhibitors:- 0.5mg/m l leupeptin (Boehringer M annheim), 0.5mg/ml protease inhibitor (Sigma), 1 mg/ml trypsin inhibitor (Sigma), 0.5mg/ml aprotinin (Sigma), and 40mg/ml bestatin (Boehringer M annheim). A fter leaving on ice for 15 minutes, 25/il of N onidet P40 (NP40, BDH) was added, the solution vortexed for 10 seconds, and microfuged for 30 seconds. An aliquot of the pellet was resuspended in PBS, and mixed with an equal volume of U nna stain (kind g ift of E. De Boer, NIMR), and viewed under the light microscope to check the integrity o f the nuclei. The nuclear pellet was resuspended in 50/il of the m odified whole cell m icroextraction b u ffer, freeze thawed 3 times, and the supernatant stored at -70°C.

2.18. ANTIBODIES

2.18.1. Polyclonal antibodies

A nti-cyclin A antibody was raised against bovine cyclin A fusion protein. A cyclin A cDNA clone (residue 26 to the 3’ non-translated end) was inserted into the T7 inducible open reading fram e of pET56, protein expressed, and recovered from inclusion bodies by SDS polyacrylamide electrophoresis and electroelution. Rabbits were injected with 200 /ig of protein at intervals of 4 weeks. This antibody was a kind gift from J. Adamczewski and T. H unt, ICRF, South Mimms.

2.18.2 Monoclonal antibodies

XZ55 is a monoclonal antibody (subclass IgG l) raised against the carboxyl terminal 60 kD polypeptide of human pRb. This antibody will im munoprecipitate and Western blot pRb from a variety of species, and preferentially interacts with the underphosphorylated form of pRb (Hu et al., 1991). K ind gift from Q. Hu and E. Harlow, Massachusetts General Hospital.

IG4E was used as a control antibody for experiments incorporating XZ55, as it is of the same subclass (La Thangue et al., 1984). K ind gift from N. La Thangue, NIMR.

A nti-SSE A l recognises a stem cell m arker protein present on undifferentiated F9 EC cells, and lost on their differentiation. The antibody is described in Solter and

Knowles, (1978) and was a kind gift from D. Solter.