Vol. 49 No. 2/2002 427–431 QUARTERLY Communication

In this study we cloned and analysed partial cDNA of tumor necrosis factor (TNF) and p75 TNF-R receptor of Syrian golden hamster (Mesocricetus auratus). We obtained a 382-bp sequence of TNF and a 148-bp sequence coding for p75 TNF-R. The primers used for the cloning were designed on the basis of inter-species homology, thus presumably can be used for cloning and analysis of TNF and p75 TNF-R genes of other mammals.

toimmune and neoplastic diseases (Aggarwal & Natarajan, 1996;Männel et al., 1993;Fiers, 1991).In order to induce cellular response TNF should bind to specific cell-surface receptors of which two species have been identified: low-affinity p55 TNF-R and high-affinity p75 TNF-R.Both receptors are present on most nucleated mammalian cells and their number depends on the tissue (De Kossodo et al., 1994;Tartaglia & Goeddel, 1992).In the present study we cloned and analysed partial cDNAs of TNF and p75 TNF-R of Syrian golden hamster (Mesocricetus auratus), an animal species useful for study on cancer, infectious diseases and other (Melby et al.,1998;Slominski & Paus, 1993).

MATERIALS AND METHODS
Male Syrian hamsters were used as a source of hamster tissues.Cells from spleen were isolated and cultured with Escherichia coli lipopolysaccharide (1 mg/ml) and then total RNA was isolated using TRI REAGENT TM (Molecular Research Center, Canada).First strand cDNA was synthesized by reverse transcription (RT) of 1 mg of total RNA with 15 U of AMV-RT (Promega Corp, U.S.A.).Primers for polymerase chain reaction (PCR) were originally designed on the basis of a homology search within the cDNA sequences of p75 TNF-R and TNF genes from various species using the program OLIGO 4.1 (National Biosciences, U.S.A.).The sequences of the primers selected were: for the p75 TNF-R gene -5¢ TCC AGC AGC AGC TCC CTA GA 3¢ and 5¢ ACG TTC ACG ATG CAG GTG AC 3¢, while for the TNF gene -5¢ AGC ACI GAA AGC ATG ATC C 3¢ and 5¢ GAA GAG AAC CTG GGA GTA G 3¢. PCR products of 420 bp for TNF and 188 bp for p75 TNF-R were isolated from agarose gel by electroelution and cloned into pGEM-T Easy vector (Promega Corp., U.S.A.) and transformed into E. coli JM109.Plasmid DNA was isolated using alkaline lysis and screened using PCR.Two independent PCR cloning experiments for each gene were performed using products obtained from different RT reactions.One clone from each cloning was chosen for sequencing, which was performed on both DNA strands.The template plasmid DNA for sequencing was prepared by using DNA Miniprep Plus kit (A&A Biotechnology, Poland), while the sequencing was performed on a Perkin-Elmer ABI PRISM 310 sequencer.

RESULTS AND DISCUSSION
The obtained 382 bp sequence of hamster TNF (GenBank accession number AF315292) and 148 bp of hamster p75 TNF-R (GenBank accession number AF315291) are shown in Fig. 1.The partial cDNA of TNF contains a sequence coding for 126 amino acids including most of the leader sequence and almost half of the sequence of the mature protein.This sequence demonstrates 89.3% homology to the appropriate part of murine cDNA, 87.7% to rat cDNA, 93.2% to Peromyscus leucopus cDNA, and 83% to human cDNA.Those values are in accord with published data pointing to high homology of TNF various species (Wedlock et al., 1996;Pennica et al., 1985).A comparison of the reported sequence with that published by Melby and co-workers (1998) indicates differences in four bases (see Fig. 1): guanine (in our sequence) instead of adenine in position 39 and cytosine instead of guanine in positions 103, 196 and 250.All of them affect the deduced amino-acid sequence.In the case of three of those differences (in positions 103, 196 and 250), the identified cytosine residues in our cDNA clones are homologous to the cytosine residues of the known TNF sequences from other species (Fransen et al., 1985;Estler et al., 1992;Crew & Filipowsky, 1992;Wang et al., 1985).Although in both known TNF sequences of members of the family Muridae adenine was identified in position 39 (Fransen et al., 1985;Estler et al., 1992), we postulate the presence of guanine in the ham-ster gene since guanine is also present in TNF sequences of other close relatives of hamster Cavia porcellus (Cavidae) and: Peromyscus leucopus (Sigmonodontidae) (Crew & Filipowsky, 1992).Of note is that P. leucopus has the highest overall homology to hamster within the TNF gene.In agreement with Melby, we found nine nucleotides unique for hamster as well as a unique 6-nucleotide deletion, which starts in position 176.
The homology of deduced amino-acid sequences (see Fig. 2), similarly to the nucleotide sequence, is high.The highest level of identity with hamster shows the sequence of P. leucopus (95.2%), while the murine, rat, guinea pig and human sequences show 89.7%, 88%, 80.2% and 73.8% of identity with the hamster sequence, respectively.Both hamster amino-acid sequences -described by Melby (Melby et al., 1998) and obtained in this work -possess features characteristic for TNFconserved b-sheets and a strongly hydrophobic 22-amino-acid membrane fragment of leader sequence (Wedlock et al., 1996).The four amino-acid differences observed between our sequence and that published by Melby most probably do not affect the function of TNF.Our results indicate the existence of structurally similar amino acids (in three cases alanines instead of glycines) and a substitution of alanine for threonine in the region where no significant homology between distant species is found.It is also possible that the differences observed may represent genetic variations between cohorts of hamsters used for our and Melby's analyses.
The sequence of hamster p75 TNF-R has not been published yet.The obtained 148-nucleotide fragment of hamster p75 TNF receptor (see Fig. 1B) covers part of the cytoplasmic domain of the receptor and codes for 49 amino acids.Homology of this sequence to the appropriate part of the murine cDNA is 83.8%, while to the human sequence -75%.These results correspond to literature data describing the highest homology between species in the cytoplasmic domain of the receptor -73% between human and mouse, while homology of the whole nucleotide sequence is 62% (Tartaglia & Goeddel, 1992).A comparison of human and mouse p75 TNF-R to the reported hamster amino-acid sequence (see Fig. 3) allows distinguishing two 11-13 amino-acid regions of high homology located at the ends of Shaded are the nucleotides differing from those present in the hamster TNF sequence published by Melby et al. (1998).Note that both sequences are given without sequences complementary to those of PCR primers.This explains the length difference between PCR products and the sequences shown.
the cloned sequence and a region of lower homology located in the middle of the presented sequence.This region contains 8 amino acids unique for hamster.
As primers used in this work were designed on the basis of homology between species, the primer pairs permit amplification of TNF and p75 TNF-R DNA not only of hamster but also of other species (see Fig. 4).Thus, the primers originally developed in this study, could be applicable to DNA amplification from species, the sequences of TNF and p75 TNF-R of which genes are known as well as unknown.Besides, the primers and the cDNAs obtained in this  Amino acids different from those present in the hamster sequence are shown, the identical amino acids were replaced by "-".Amino acids unique for the hamster sequence are bolded.The sequences used for comparison were 2. m59378 (Goodwin et al., 1991), 3. m55994 (Kohno et al., 1990), according to their GenBank accession numbers.
work can serve as specific molecular probes in studies on the biology of TNF and its receptors in hamster models of infectious and neoplastic diseases.PCR products (TNF -420 bp for hamster and 426 bp for other species; p75 TNF-R -188 bp) and molecular weight markers: pKO3/HinfI and pUC19/MspI were separated on 1% agarose gels and stained with ethidium bromide.Reaction mixture contained (conditions for p75 TNF-R amplification different from those for TNF are in parentheses): 1 x Taq DNA polymerase buffer, 2.5 mM MgCl 2 , 2 (1) U Taq DNA polymerase (Promega Corp., U.S.A.), 50 (100) pmol each of forward and reverse primers, 200 mM dNTPs (Promega Corp., U.S.A.) and redistilled water to total volume 50 ml.PCR parameters were as follows (values for p75 TNF-R are given in parentheses): initial denaturation at 95°for 4 min; 33 cycles of: 95°for 30 s (1 min), 54.6°C for 30 s (58.8°C for 1 min), 72°C for 1 min (2 min); final extension at 72°C for 5 min (7 min).

Figure 1 .
Figure 1.Nucleotide sequence of partial cDNA of Syrian hamster TNF (A) and p75 TNF-R (B).

Figure 2 .
Figure 2. Comparison of amino-acid sequence of the obtained fragment of hamster TNF with the previously published hamster sequence and with the sequences of other species.

Figure 3 .
Figure 3. Alignment of hamster, mouse and human amino-acid sequences of p75 TNF-R.