3.1 EVOLUCIÓN DE LAS INSTITUCIONES PÚBLICAS DE VIVIENDA EN LA
3.1.1 COMPAÑÍA DE VIVIENDA PROVINCIAL (COVIPROV)
3.1 INTRODUCTION
All members of the Eph family were initially identified as orphan receptors (Bennett et a i, 1995; Harai et ai, 1987; Maisonpierre et a l, 1993). When the first ligands were identified, it was found that they were members of a novel gene family. Sequence alignments of these so-called ephrins (ephrin-Al, ephrin-A2, ephrin-A3 and ephrin-Bl), showed that they shared conserved regions including four conserved cysteine residues (Cheng and Flanagan, 1994; Davis et al, 1994). The ephrin sequences also indicated that ephrin-Bl contained a transmembrane domain whereas the carboxy terminal ends of ephrin-Al, ephrin-A2 and ephrin-A3 encoded a glycosylphosphatidylinositol (GPI) linkage motif. Using this sequence information, I designed primers for use in a PCR- based strategy, based on regions of high homology between these ephrins.
This chapter describes the identification of ephrin-A ligands in the early Xenopus embryo by this PCR strategy. I will analyse the sequence data obtained from the PCR clones and compare them with ephrins previously cloned in other vertebrate species.
3.2 RESULTS
3.2.1 RT-PCR Procedure
The primer design was based only on the sequences of ephrin-Al, ephrin-A2 and ephrin- A3 since the ephrin-Bl sequence is divergent and a primer could not be designed that was long enough to accommodate amplification of both subclasses.
Three degenerate oligonucleotide primers were designed, two 5' and one 3', suitable for use in an RT-PCR based strategy. These primers were used to amplify a conserved region of 165 nucleotides in length, specific for members of the ephrin-A class. The region chosen for the screen is shown in Fig. 3.1. The amino acid sequences corresponding to the primers are shown below with their nucleotide sequences detailed in Chapter 2.
5' (V/L)DI(I/Y)CP Oligo 1 5' (V/L)DI(IA")CPHY Oligo 2
3' EKFQR(F/Y) Oligo 3
Figure 3.1
The primers chosen to amplify ephrins in Xenopus
The regions of homology between ephrin-Al, ephrin-A2 and ephrin A3 which were used to create degenerate oligonucleotide primers for the RT-PCR procedure are shown.
The arrows above the sequence indicate the 5' and 3' ends of the primer sequences. The sequence motifs are in bold. The boxed amino acids indicate sequence expected in ephrin clones. The conserved cysteine residues are high-lighted in red.
E P H R I N - A 1 T I H V Q L N DI I E P H R I N - A 3 TVQVNVN DI Y E P H R I N - A 2 T V E V S I N DI Y E P H R I N - B I V I Y P K I G 1 DII C O N S E N S U S T I . V . . N d|y l DI Y CPHYEDH S V . . . A D A A M PHYNSS GAGPGPGGGA PHYGAP L . . P . PAERM P R A E ...AGRPY E
Es.
ER AG . . . . E Q Y . L Y L V . YV YY VLY^/VLY LYPVNY KLY VEH SR VRP E P H R I N - A 1 EPH R 1N -A 3 EPH R 1 N -A 2 E P H R I N - B l EEYQL NGYRT EGHAS EQAAA C O N S E N S U S E . Y . . 2 P QS KDQVRWCCNR P XfAXS QGFKRWECNR P pHRQ RGFKRWECNR P STVL DPNVLVTCNR P . . . S . . . VRW. CNR P SAKHGPE B A P H S P I AAPGGPLKF SEKF' E Q E . . . I . A . H . P I KL SEKFQ KF S E K F ‘ RF TIKFQ KF SEKFQ R F T P F RYSAF L F T P F E F S P N R F S P FEach primer was also designed to incorporate a restriction site, such that the 5' primers, Oligo 1 and 01igo2, included the restriction site for EcoRI and the 3' primer, Oligo 3, a restriction site for BamHl. These restriction enzyme sites would increase the likelihood that the subcloned EcoRl-BamHl digested PCR products contained the correct 5' and 3' primers. With the addition of these restriction enzyme sites and the primers, the amplified PCR products would be approximately 212bp in size.
Total RNA was extracted from Xenopus embryos at stage 10, just after mesoderm induction and the beginning of gastrulation, and at stage 17, when neurulation is almost complete. The RNA was reverse transcribed using both oligo dT and random hexamer primers. In initial experiments PCR was carried out using a final MgCE concentration of 3 mM, and an annealing temperature Tm (melting temperature) of 47°C. The Tm was calculated as indicated below, where the nucleotides are those encoding the primers.
4°C (G/C) + 2°C (ATT) = Tm
I was not able to see any PCR products on an agarose gel run with the resultant PCR products. It was possible either that the concentration of the MgCl% was not optimal or that the annealing temperature had to be changed. To address this problem, PCR reactions were set up at three final concentrations of MgCE (4.5 mM, 3 mM and 1.5 mM) and at each of the following annealing temperatures: 42°C, 47°C and 51°C. PCR products of 212 bp in size were obtained at all three annealing temperatures when the MgCL final concentration was 1.5 mM. Therefore, the MgCL concentration had been ] sub-optimak
The PCR reaction products were run on a 2% agarose preparative gel. DNA running at a position equivalent to 212 bp was extracted. A representative PCR gel shown in Fig. 3.2 indicates the close migration of the molecular weight bands representing 201 bp and 220bp. PCR products present at, or near, the position of these migrated markers, were excised. This DNA was digested using the restriction enzymes EcoRI and BamHl and subcloned into similarly cut and de-phosphorylated pBluescript (KS+) vector. De phosphorylation was used to decrease the occurrence of vector-vector re-ligation.