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Molecular cloning, tissue distribution and sequence analysis of complete glucokinase cDNAs from gilthead seabream (Sparus aurata), rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio)

“…In order to obtain a full-length cDNA sequence data for GK from the three teleosts, we used an established strategy called Rapid Amplification of cDNA Extremities (RACE-PCR). This was possible because we already had the partial sequence of these cDNAs [19].…”

“…The 5' and 3' cDNA extremities were determined by the RACE-PCR method as detailed in the manufacturer's notice (Boerhinger, Roche Molecular Biochemicals, Germany). The teleost GK specific primers were designed from the partial sequence data of the same species previously obtained in our laboratory [19] (Table 1). Using the reverse transcription system, cDNA was synthesized by incubating 1μg of polyA mRNA from fish livers with 5 AMV reverse transcriptase for 1h at 42°C using either the oligodT primer (3' Race) or a species-specific GK primer (5' Race) ( Table 1).…”

“…Samples of 20 μg of total RNA samples were submitted to electrophoreses on 1% agarose gels containing 5% formaldehyde and capillary transferred onto nylon membrane (Hybond-N + , Amersham, UK). Membranes were hybridized with [ 32 P] DNA specific for GK sequences labeled by random priming (Stratagene, USA) [19] (Genbank accession numbers AF053330, AF053331 and AF053332 for gilthead seabream, rainbow trout and common carp GK related probes respectively). Membranes were also hybridized with a carp 16 S ribosomal RNA probe (Genbank accession number MICCCG) to check the inter-sample variation in loading.…”

“…Literature data in fish on the existence of a functional GK-like enzyme and on the induction of GK expression by dietary carbohydrates have been rather contradictory [13,14,15,16,17,18]. We recently reported isolation and characterisation of hepatic cDNA sequences from three cultured teleosts which are homologous to a portion of mammalian GK sequences [19]. These three teleosts differ in their capacity to utilise dietary carbohydrates : an omnivorous fish, namely common carp, able to use efficiently high levels of dietary carbohydrates and two carnivorous species, rainbow trout and gilthead seabream, less tolerant to dietary carbohydrates [8,9].…”

“…By annealing 2µg of total RNA with 1µg of random primers and incubating with AMV reverse transcriptase (Invitrogen, USA) for 1h at 42°C, total cDNA was synthesized. Using specific primers derived from the previously acquired partial GK cDNA sequences [19] (Table1), GK-specific cDNA fragments were synthesized. At this end, a 35 cycle PCR reaction was carried out in a final volume of 25 μl containing 1.5 mM MgCl 2 , 4 pmol of each primer, 2 μl total cDNA and 1 U of Taq polymerase (Boerhinger, Roche Molecular Biochemicals, Germany) with an annealing temperature of 51°C for common carp and gilthead seabream, or 55°C for rainbow trout.…”