El Factor estructural y los límites de la representación Aimara

RT-PCR

Reverse transcriptase-PCR (RT-PCR) was carried out in two stages: reverse transcription to produce cDNA from an RNA sample and then PCR to amplify a specific transcript.

Reverse transcription

The reaction was carried out with 1 |ig of total RNA. cDNA synthesis was initiated using random primers, which were annealed by heating the sample to 70°C for 10 min and cooling on ice for 2 min. The first strand synthesis reaction contained 500 pM dNTPs, 10 mM DTT, 40 U RNasin, 200 U superscript II reverse transcriptase (GIBCO BRL) and buffer. The mixture was incubated at 42°C, first for 2 min before addition of enzyme and then for 1 hour.

PCR

1 pi of the above reaction containing first-strand cDNA was used as the template in a PCR reaction, essentially as described above (see PCR) using Pfu polymerase.

DNA Sequencing

DNA was sequenced by the dideoxy-mediated chain-termination method using the T7 Sequenase (version 2.0) kit from Amersham. 5 pg of template DNA was denatured by adding 0.1 volume of 2 M sodium hydroxide, incubating at 37°C for 5 min. The mixture was neutralised by adding 0.1 volume of 5 M ammonium acetate and the DNA

precipitated by adding 4 volumes of ethanol, mixing, cooling on dry ice for 5 min and centrifuging at 4°C for 5 min. After washing the pellet with 70% ethanol, the DNA was resuspended in water. The specific sequencing primer was annealed to the denatured DNA by heating to 70°C and cooling to 3TC over 20-30 min.

Chapter 2. M aterials & M ethods

The labelling reaction was carried out at room temperature for 2-5 min. The reaction contained the denatured DNA with the annealed primer, 1 mM DTT, 3.25 U T7

Sequenase polymerase and 0.37 MBq p^S]-ATP (37 TBq/mmol). The mixture was then split into 4 and a quarter was added to each of the four wells, each containing one of the four dideoxy nucleotides, and incubated at 37°C for 5 min. The reaction was stopped by adding 4 pi of STOP solution (formamide loading buffer). The four

reactions were then run on a 6% acrylamide/7 M urea gel until the xylene cyanol in the loading buffer (STOP solution) had travelled 75% of the gel length. The gel was then fixed in 10% methanol/10% acetic acid for 10 min, dried for 30 min and exposed to X- ray film at -80°C with an intensifier screen.

In vitro transcription

Synthetic capped mRNA for microinjection was produced by in vitro transcription reactions containing commercially available RNA polymerases (SP6, T3 and T7; Roche). Plasmid DNA encoding the specific gene was linearised with a restriction enzyme site at the 3' end of the gene (either blunt-ended or producing a 5' overhang), cleaned by phenol/chloroform extraction and precipitated to produce the template. The constructs used in this thesis are shown below (Table 2.1).

Reactions contained 5 pg of linearised template, 10 mM DTT, 100 U RNasin, 1 mM each of ATP, CTP and UTP, 100 pM GTP, 500 pM cap structure [m7G(5')ppp(5')G], 50 U of RNA polymerase plus 1 x transcription buffer. This was incubated for 30 min at 37°C, when more GTP was added to bring the concentration up to 500 pM and the reaction was incubated at 37°C for a further 1 hour. The mixture was then treated for 30 min at 37°C with 50 U RNase-free DNase I to remove the template, phenol/chloroform extracted and the supernatant cleaned further by the use of a CHROMASPIN-100 (Clontech) colunrn to remove unincorporated nucleotides. The RNA was then concentrated, if necessary, by adding 0.5 volumes ammonium acetate, 2-3 volumes ethanol, cooling on dry ice for 5 min and centrifuging at 13,000 rpm to precipitate it.

Chapter 2. M aterials & M ethods

Table 2.1. Constructs used as templates for in vitro transcription reactions

Gene Plasmid Linearisation site Polymerase Reference

Activin pSP64T-m|jA Sma I SP6 (Albano et al., 1993)

P -galactosidase pSP6nucP0al Xho I _{SP6 (Smith and Harland, 1991)}

derrière pCS2^-derrière Not I SP6 (Sun et al., 1999)

Eomesodermin pSP64T-Eomes-HA Sal I SP6 (Conlon et al., 2001)

FASTI pFTX9-XlFastl Xba I T7 (Howell et al., 1999)

VegT pSP64T-VegT-HA Sal I SP6 (Conlon et al., 2001)

VegT-GR pSP64TBX-VegT-GR Sal I SP6 This thesis

Xbra pSP64TBX-Xbra-HA Sal I SP6 (Tada et al., 1997)

Xnr2 pXnr2 Sma I SP6 (Jones et al., 1995)

In vitro translation

Proteins were produced by in vitro translation reactions, using rabbit reticulocyte lysate (Promega). Reactions contained 400 ng synthetic capped RNA, 8 |il reticulocyte lysate, 20 U RNasin, 50 pM amino acids (-Met), and 0.185 MBq [^^S]-Met (37 TBq/mmol). The reaction was incubated at 30°C for 70 min and then labelled proteins were separated on a 10% SDS-PAGE gel. Gels were run at 150 V for 45 min, fixed in 10%

methanol/10% acetic acid, dried and exposed to X-ray film at room temperature.

Gel mix 30% acrylamide Tris-HCl buffer 10% SDS 10% APS TEMED H2O RESOLVING 3.3 ml 2.5 ml (1.5 M pH 8.8) 0.1 ml 0.1 ml 0.004 ml 4.0 ml STACKING 1.6 ml 1.25 m l(lM p H 6.8) 0.1 ml 0.1 ml 0.01 ml 6.9 ml

Chapter 2. M aterials & M ethods

Site-directed mutagenesis

All mutagenesis was performed using a two-step PCR-based approach.

Oligonucleotides were designed and contained the mutations to be made and at least 15 base-pairs of sequence on either side (5' and 3') to ensure efficient hybridisation (Fig. 2.1 P2 and P3). Two overlapping products were produced using these primers and primers outside the insert region (Fig. 2.1 P1 and P4). These products were then pooled and a further round of PCR using just the most 5' and 3' primers (Fig. 2.1 P1 and P4) was used to produce the final mutagenised insert (Fig. 2.1),

ATbs primers

M l GGA T G A GGG A C T G T T GGC A T C G C T C T C C T G A C T C C A C

M l < - C T C C A C T C A C C A CA C C C A T C C CA A C A C T C C C T C A T C C

M2 C T C C A T C A C AAC A CA C A A C C C T A T CA C A T A C C T C A T T C

M2 < - CA A T C A C C T A T C T C A T A C G C T T C T C T C T T C T C A T C C AC

Area spanning the T-box site is underlined. The consensus T-box site is the 8 base-pair sequence T C A C A C C T as defined by Conlon et al. (2001). Base changes are in bold. AFAST primers

AF-> C C A A T C C C T C T A A T T AAG A T C CAC A T C T A T A T T C T C T C C A T C

AF<- A T C CAC A C A A T A T A G A T C T C C A T C T T A A T T A C A C C C A T T

Fast sites are underlined. The Fast site is an 8 base-pair site with the consensus

Chapter 2. M aterials & Methods