CLONING TWO PepT1 cDNA FRAGMENTS OF COMMON CARP, CYPRINUS CARPIO (ACTINOPTERYGII: CYPRINIFORMES: CYPRINIDAE)

1 Division of Ichthyobiology and Fisheries, Faculty of Animal Science, Warsaw University of Life Sciences, Warsaw, Poland 2 Division of Molecular Cytogenetics, Faculty of Biotechnology and Animal Science, West Pomeranian University of Technology, Szczecin, Poland 3 Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, via Provinciale LecceMonteroni, Lecce, Italy 4 School of Environment and Natural Resources, Ohio State University, Columbus, Ohio, USA


INTRODUCTION
In genome sequencing of prokaryotes and eukaryotes two strategies are applied. The first, involves sequencing the entire genome. The second, and less common, approach involves sequencing specific parts of the genome, usually a single gene. However, irrespective of the strategy applied, the selection of organisms for genomic research is a most important task. The most commonly applied criterion regulating such selection, besides that of "model organism" (e.g., mouse, or rat), is consideration of the biological and commercial importance of an organism, a consideration aimed at guaranteeing that the genomic results would be practically applied. This criterion is relevant to common carp (Cyprinus carpio), for which small sections of nuclear and mitochondrial genomes have been sequenced, but for which specifics regarding genome structure, regulation of gene expression, and molecular determination of features relevant to commercial interest still need to be elucidated. Therefore, partial sequencing of the common carp PepT1 gene (called also SLC15A1 -SoLute Carrier 15 A1), coding for an oligopeptide membrane transporter protein was assumed meaningful for some reasons: its expression is related to digestive tract differentiation and development, digestion physiology, and nutrient transport. Up to now the PepT1 sequences were described among other fish: zebrafish, Danio rerio (see: Verri et al. 2003), icefish, Chionodraco hamatus (see: Maffia et al. 2003), and Atlantic cod, Gadus morhua (see: Amberg et al. 2008). These issues are of key importance for optimization of stocking material pre-rearing technique development. With the above in mind, we herein report research findings from a study designed to sequence the PepT1 (SLC15A1) in common carp.

MATERIALS AND METHODS
PCR-based cloning of cDNA fragments encoding for carp PepT1. Common carp intestine was isolated from 6-week-old fish (5 individuals) killed by overdose immersion in the anaesthetic 3-aminobenzoic acid ethyl ester (MS-222 Sigma-Aldrich, Poznań, Poland). Following euthanasia, the entire length of the intestine was removed. Tissue was briefly rinsed in ice-cold saline solution (1.1% NaCl), immediately fixed in RNA later (Ambion, Inc., Austin, TX, US), and stored at -80°C until use.
Total RNA was extracted from the RNAlater-stored intestine using Trizol Reagent (Invitrogen, Carlsbad, CA, US) according to the manufacturer's instructions. Reverse transcription was performed at 50°C for 60 min using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, US), oligo(dT) 12-18 primer and 5 µg total RNA following the manufacturer's instructions. Two microliters of resulting cDNA product was used to perform PCR with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, US) in the presence of zebrafish-specific primer pairs designed according to the nucleotide sequence of zebrafish PepT1 cDNA (zfpept1; GenBank acc. no. AY300011) . Two different primer pairs were synthesized commercially (Proligo France SAS, Paris, France) and used to amplify and clone two conserved fragments of carp PepT1 cDNA. The primer pair zfPepT1F1 (5'-TGTATCTGTC--TATAGTGTAC-3', identical to nucleotides 387-406 of zebrafish PepT1 cDNA) -zfPepT1R1 (5'-CAAATGCT--GCCACACAC-3', the reverse complement of nucleotides 556-572 of zebrafish PepT1 cDNA) was expected to amplify a 187 bp fragment and the primer pair zfPepT1F2 (5'-TCAGTTTGAAGATCACCAGG-3', identical to nucleotides 580-599 of zebrafish PepT1 cDNA) -zfPepT1R2 (5'-AGTATAGGGTTCAC--GATCTG-3', the reverse complement of nucleotides 1109-1128 of zebrafish PepT1 cDNA) was expected to amplify a 549 bp fragment. After a denaturing step at 94°C for 2 min, PCR amplification was carried out for 35 cycles, with denaturation at 94°C for 30 s, annealing at 50°C for 60 s, and extension at 72°C for 60 s, and a final extension at 72°C for 7 min. RT-PCR products were separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. PCR amplification products of the expected size were eluted using the QIAquick Gel Extraction kit (Qiagen, Chatsworth, CA, US) and subsequently cloned in the pCRII-TOPO vector (TOPO TA Cloning, Invitrogen Carlsbad, CA, US). The plasmid clones containing the isolated cDNA fragments were sequenced using universal primers.

Table 1
Nucleotide sequence of the first fragment of common carp, Cyprinus carpio, PepT1 gene divided into codons, and inferred primary protein structure Yellow-conservative sequences among fish species of PepT1 gene; Blue-conservative sequences among vertebrate species of PepT1 gene.

RESULTS
Two PepT1 cDNA fragments from common carp were cloned, sequenced and translated into their potential amino acid sequence (Tables 1, 2).
Equally high similarity at the DNA level is also observed for mammals and birds; however, only two species of the latter were compared. Inferred amino acid sequences from this fragment spanned from transmembrane domain 3 (TM 3) to TM 4 and exhibited very high (86%) similarity to the zebrafish PepT1 amino acid sequence (Tables 2, 4). The same refers to the taxa mentioned above.
Similarly, the second cDNA fragment exhibited 87.5% similarity to the zfPepT1 gene fragment and 63.5 % similarity among the teleosts. High level of homology of this fragment was observed also in GenBank PepT1 sequences for birds and mammals, 97.5 and 63%, respectively (Tables 2, 3). The inferred amino acid sequence from this fragment spanned from TM 5 to the start of TM 8 and exhibited 92% similarity to the zebrafish PepT1 amino acid sequence (Table 5), 76.5% among other fish, bird (97.5) and mammal (80.5) species.   The partial nucleotide sequences of the carp PepT1 exhibited highest sequence similarity to the zebrafish PepT1. In particular, the first cDNA gene fragment sequence exhibited 84% similarity to zfPepT1 and over 64% with respect to the three teleosts for which sequences were made available (Table 3).

Table 4
Comparison of amino acid sequence of PepT1 protein for various vertebrates available from the GenBank database-fragment 1 It is noteworthy that considerable conservatism at the level of primary protein structure was observed mainly for transmembrane domain regions (Table 6), particularly in TM 5, while in connecting loops homology was much lower.

DISCUSSION
Peptide transporters (PEPT) are membrane proteins responsible for selective transport of small peptides across the intestine enterocyte membranes (Chen et al. 2005). Among them, PepT1 is present in the small intestine villi and is of key importance for absorption of protein hydrolysis products, particularly dipeptides and tripeptides. The PepT1 protein in higher vertebrates shows similar length to each other, usually about 700 amino acid residues (708 in human, 707 in rat, and 709 in mouse,) while in lower vertebrates it tends to be longer. The difference relates rather to the length of loops connecting transmembrane domains than the length of the domains themselves (Liang et al. 1995, Miyamoto 1996, Fei et al. 2000. Although no crystallographic structure of PepT1 protein is known, a probable model of PepT1 protein was created (Abramson et al. 2003, Huang et al. 2003) using appropriate software, and crystallography of similar transporter proteins of E. coli LacY (crystallized bound to the substrate), and GlpT (crystallized without a substrate). PepT1 shows an α-helix structure, and consists of 12 functional transmembrane domains, nonlinearly distributed within the cell membrane. It seems that a hydrophilous cation (H + ) transmembrane channel exists, through which dipeptides, tripeptides and free amino acids are transported (Meredith and Price 2006). Among the 12 domains, seven are directly involved in channel structure (domains 1, 3, 5, 7, 8, 9, and 10). In the present study, the in silico analysis revealed that domains 5, 6, and 7 were the most conservative, while connecting regions (loops) (except for the one connecting domains 7 and 8) were more variable. Particularly domain TM 5, which is responsible for regulation of the rate of substrate transport through the cation channel, showed high level of conservatism (72%). It is similarly to the domains 6 and 7, which participate in initiation of substrate binding, and regulate the rate of their flow (61% and 63%, respectively). It is worth mentioning that an important role of some amino acid radicals was observed in other vertebrates, such as Y167, R282, or W294, substitution of which considerably disturbed biological activity of PepT1 protein (Bolger et al. 1998).

Table 6
Similarity of amino acid sequences in various species with division into transmembrane domains and connecting loops In the fragments of PepT1 protein analyzed in the present study these sites are identical as in other species. Concluding, we can assume that high homology of PepT1 gene at the DNA level, and conservative primary structure of PepT1 protein probably reflect PepT1 conservative function, the pattern of expression, or PepT1 level corresponding to the internal environment conditions. The obtained partial sequence of common carp PepT1 should allow to test this hypothesis in future studies.