Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor

Abstract

Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor 111 RNA isolation, RT-PCR and RACETotal RNA from platelets, cells or tissues was purified according to the M-MLV Reverse Transcriptase system (Invitrogene) using the random primers pd(N) 6 -5'-PO 3 NA + Salt (Pharmacia Biotech). RNA samples were kept at -80ºC after controlling the quality on a denaturing agarose gel. 5 µg RNA, resuspended in 9.5 µl water, were heated for 3 min at 65ºC in the presence of random hexamers (7.5 µM final concentration), and then cooled in ice. RNA was reverse transcribed using 1 µl Moloney murine leukemia virus reverse transcriptase (200 units/µl) (GibcoBRL) for 1 h at 42ºC in a final volume of 20 µl containing 4 µl of 5X reverse transcriptase buffer, 0,5 µl ribonuclease inhibitor (50 U/µl) (Roche), 1 µl 20 mM dNTP (Pharmacia) and 2 µl 0.1 M dithiothreitol. After 10 min at room temperature, 1 h at 42 º C, and 10 min at 95ºC, DEPC H 2 O were added until a final volume of 100 µl. 2 µl of this mixture were subjected to PCR using 1µl Tth DNA polymerase (1U/µl) (Biotools) and 2,5 µl each CD41-specific primer (20µM) (see Table 1) in a final volume of 50 µl containing 5 µl 10x buffer, 2 µl MgCl 2 (50 mM), and 8 µl dNTP MIX (1,25 mM each) (Biotools). The amplification consisted in 35 cycles of PCR and each cycle consisted of incubations at 94ºC for 1 min, TmºC for 1 min, and 72ºC for 1 min. The amplifications were electrophoresed on 1% agarose/1X TAE gel. RT-PCR on RNA18S cDNA was used as a control. For RACE (Rapid Amplification of cDNA Ends), 1 µg total RNA from platelet was used to reverse-transcribe using 1 µl Moloney murine leukemia virus reverse transcriptase (200 units/µl) (GibcoBRL) for 1 h at 42ºC in a final volume of 20 µl containing 4 µl of 10X reverse transcriptase buffer, 1,0 µl ribonuclease inhibitor (50 U/µl) (Roche), 4 µl 2,5 mM dNTP (Pharmacia) and 2 µl 3' RACE ADAPTER (20 µM) in a final volume of 20 µL. 3' CD41 cDNAs were obtained by PCR using a specific porcine CD41 primer and the anchor primer provided in the kit (Table 1). DNA sequencing and sequences analysisSequencing was performed using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on a thermal DNA cycler GeneAmp PCR System 2400 (Applied Biosystems, Foster City, CA, USA), according to the instructions of the manufacturer, and analysed on an ABI PRISM 3100 Sequencer (Applied Biosystems, Foster City, CA, USA). Porcine CD41 sequence has been deposited at GenBank under number JF808665. Sequences were analyzed using the analysis software LaserGene (DNAstar, Londo, UK) and the analysis tools provided by the expasy web site <http://www.expaxy.org>. Primers design was performed with Oligo 6 (MBI, Cascade, CO, USA) and Amplify 3 (<http://www.engels.genetics.wisc.edu/amplify/>). Multiple alignment among CD41 peptide sequences from Sus scrofa (GenBank accession no. JF808665 ), Homo sapiens (GenBank...