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IRUCAA@TDC : Sac I Restriction Fragment Length Polymorphism (RFLP) related to the human CST2 gene

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(1)Title. Author(s) Journal URL. Sac I Restriction Fragment Length Polymorphism (RFLP) related to the human CST2 gene Minaguchi, K; Kiriyama, T; Saitoh, E; Isemura, S; Sanada, K Bulletin of Tokyo Dental College, 43(1): 41-44 http://hdl.handle.net/10130/302. Right. Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/.

(2) 41. Bull. Tokyo dent. Coll., Vol. 43, No. 1, pp. 41⬃44, February, 2002. Short Communication. SAC I RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) RELATED TO THE HUMAN CST2 GENE KIYOSHI MINAGUCHI, TATESHI KIRIYAMA, EIICHI SAITOH*, SATOKO ISEMURA** and KAZUO SANADA*** Department of Forensic Odontology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan * Department of Oral Biochemistry, School of Dentistry at Niigata, The Nippon Dental University, 1-8 Hamaura-cho, Niigata 951-8580, Japan ** The Nippon Dental University Junior College at Niigata, 1-8 Hamaura-cho, Niigata 951-8580, Japan *** Department of Biochemistry, School of Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi-cho, Chiyoda-ku, Tokyo 102-8159, Japan Received 7 January, 2002/Accepted for Publication 4 February, 2002. Abstract Restriction Fragment Length Polymorphism (RFLP) related to cystatin gene (CST) family was detected in the Japanese population by using restriction enzyme Sac I. A polymorphic site, located at 0.9 kb from the 3⬘ end of the CST2 gene, revealed a two allele polymorphism with band sizes of 3.5kb and 8.3kb by hybridization with probe including exon 2 of the CST1 gene. The gene frequencies in the Japanese population were 0.326 for 3.5 kb allele and 0.674 for 8.3kb allele (n⳱86). The phenotypes of the polymorphism showed no association with the previously reported electrophoretic cystatin SA protein phenotypes22). Key words:. Type 2 cystatin gene family— CST2— RFLP— SNP— Salivary cystatin SA — Polymorphism. The human type 2 cystatin gene family is a multigene family composed of seven members localized on chromosome 20p11.22,9,10,12,21,22,24) and five of these genes (CST1-5) produce proteins which are secreted in human saliva1,3,11,14–16). The presence of genetic polymorphisms in the human type 2 cystatin gene family has been demonstrated in the CST3, CST5, and CST2 genes4–8,13,17,23), and a number of single. nucleotide polymorphisms (SNPs) related to the type 2 cystatin gene family have been submitted in the JSNP database (the database of Japanese single nucleotide polymorphism) with recent progress on genomic sequencing: 1 SNP for CST1, 4 for CST3, 4 for CST4, 5 for CST5. In this paper, we report a RFLP related to the CST2 gene, and also show a relationship with the previously reported cystatin SA 41.

(3) 42. K. MINAGUCHI et al.. protein polymorphism23). After informed consent was obtained, blood and saliva samples were collected from 86 subjects, and genomic DNA was obtained from the blood by usual Phenol/Chloroform extraction method. Genomic DNA was digested with Sac I, electrophoresed on a 0.8% agarose gel, and transferred to nylon membranes (NY-2N, Schleicher & Schuell). A 0.86 kb NcoI-NcoI fragment including exon 1 of the CST1 gene and a 0.6 kb EcoRI-NcoI fragment including exon 2 of the CST1 gene (Fig. 2) were used as probes for hybridization20). The fragments were labeled with [32P]-dCTP by nick translation. The filters were washed twice with 3X SSC including 0.5% SDS at 68°C for one hour and once with 0.3X SSC including 0.5% SDS at 68°C for one hour. The filters were exposed two days on Kodak X-OMATTMAR radiographic film. Salivary cystatin protein polymorphism was typed as previously described23). By Southern hybridization using exon 1 and exon 2 probes, no difference was observed with the exon 1 probe, but a distinct difference was observed in 8.3 kb and 3.5 kb bands using the exon 2 probe (Fig. 1). Pedigree analyses of three families confirm that these three types follow Mendelian inheritance. Among the 86 samples, 37 (39.1 expected) possessed a 8.3 kb band (genotype 8.3/8.3), 42 (37.8 expected) possessed two bands of 8.3 and 3.5 kb (genotype 8.3/3.5), and 7 (expected 9.1) possessed a 3.5 kb band. The gene frequencies for the 8.3 kb allele and 3.5 kb allele obtained in the Japanese population were 0.674 and 0.326, respectively. There was a good agreement between observed and expected numbers of genotypes assuming a Hardy-Weinberg equilibrium (␹2⳱1.06, d.f.⳱1, 0.3⬍p⬍0.5). Fig. 2 shows a restriction map of type 2 cystatin gene family reported by Saitoh et al.18,19) including data by Freije et al.11). Among these genes, the CST2B gene had been proposed to be an allele of the CST2 gene18). If the exon 2 probe hybridized to all known genes reported up to date, each gene should generate a 2.7 kb band for CST1, a 3.5 kb band for CST2, a band larger than 5.6 kb for CST2B, a 2.5 kb band for CST3, a 2.8 kb band for CST4, a. Fig. 1 Southern blot hybridization of Sac I digest of human genomic DNA from 6 individuals showing constant band, at 2.8 kb and polymorphic bands at 3.5 and 8.3 kb (indicated on the left). Channel 1; 8.3/3.5, channel 2; 8.3/ 8.3, channel 3; 3.5/3.5, channel 4; 8.3/8.3, channel 5; 8.3/3.5, channel 6; 8.3/3.5. band larger than 2.84 kb for CST5, a 1.95 kb band for CSTP1 and a 2.84 kb band for CSTP2. Pairwise comparisons of the nucleotide sequences between the exon 2 EcoRI/ NcoI probe and six CST genes showed homologies of 100% for CST1, 93% for CST2 (or CST2B) and CST4, 68% for CST3, 65% for CST5, 61% for CSTP1, and 62% for CSTP2. From these observations, it was considered that the most intensified 2.8 kb band was a mixture derived from CST1 and CST4, and the 3.5 and 8.3 kb bands with similar intensities were derived from CST2 and CST2B, respectively, because the homology between the exon 2 probe and these three genes was high (93–100%); the 3.5 kb band matched the expected size derived from CST2, and the 8.3 kb band could only be derived from CST2B. The CST2 gene contained in the sequence from clone XXyac-60D10 on chromosome 20 (Accession no. AL591074) has two Sac I sites spanning 8.2 kb and including.

(4) 43. SAC I RFLP OF THE HUMAN CST2 GENE. Fig. 2 The restriction map of the type 2 cystatin genes modified from the report by Saitoh et al.18) including the data for CST511) and CSTP219). The CST1-5 genes are functional genes and the CSTP1 and CSTP2 are pseudogenes. The abbreviations of the restriction enzymes are as follows; H (Hind III ), N (Nco I ), E (Eco RI ), S (Sac I ) and P (Pst I ). Positions of exons are indicated by solid boxes. “Exon 1 Frag.” and “Exon 2 Frag.” correspond to the regions from which probes were prepared.. Table 1. Relationship between Sac I RFLP genotypes and salivary cystatin SA (CST2) genotypes. Sac I RFLP genotype Cystatin SA (CST2) genotypes 8.3/8.3 8.3/3.5 3.5/3.5 CST2*1/CST2*1 CST2*1/CST2*2 Total. 27 7 34. 32 6 38. 6 0 6. Total 65 13 78. ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research (C) and (A) from the Ministry of Education, Science and Culture, Japan.. REFERENCES. exon 2 and exon 3, which coincides with CST2B and supports the present observation. We have already reported a salivary protein polymorphism derived from the CST2 gene13,23). This polymorphism is caused by two point mutations in exon 2 and exon 3 of the CST2 gene. When 78 samples were compared with respect to the association of phenotypes between salivary cystatin SA protein polymorphism and Sac I RFLP, there was no significant association (Table 1). Thus these polymorphisms might have occurred independently. It will be possible to apply the present polymorphisms of the CST2 gene to forensic studies and linkage studies on chromosome 20.. 1) Abrahamson, M., Barrett, A.J., Salvesen, G. and Grubb, A. (1986). Isolation of six proteinase inhibitors from urine. J Biol Chem 261, 11282–11289. 2) Abrahamson, M., Islam, M.Q., Szpirer, J., Szpirer C. and Levan, G. (1989). The human cystatin C gene (CST3) mutated in hereditary cystatin C amyloid angiopathy is located on chromosome 20. Hum Genet 82, 223–226. 3) Al-Hashimi, I., Dickinson, D.P. and Levine, M.J. (1988). Purification, molecular cloning, and sequencing of salivary cystatin SA-I. J Biol Chem 263, 9381–9387. 4) Balbin, M. and Abrahamson, M. (1991). SstII polymorphic sites in the promoter region of the human cystatin C gene. Hum Genet 87, 751–752..

(5) 44. K. MINAGUCHI et al.. 5) Balbin, M. and Abrahamson, M. (1992). PCR assay for a polymorphic DdeI site in the promoter region of the human cystatin C gene. Hum Genet 88, 710. 6) Balbin, M., Freije, J.P., Abrahamson, M., Velasco, G., Grubb, A. and Lopez-Otin, C. (1993b). A sequence variation in the human cystatin D gene resulting in an amino acid (Cys/Arg) polymorphism at the protein level. Hum Genet 90, 668–669. 7) Balbin, M., Grubb, A. and Abrahamson, M. (1992). Demonstration of sequence variations in the promoter region of the human cystatin C gene. J Biol Chem 373, 471–476. 8) Balbin, M., Grubb, A. and Abrahamson, M. (1993a). An Ala/Thr variation in the coding region of the human cystatin C gene (CST3) detected as a SstII polymorphism. Hum Genet 92, 206–207. 9) Dickinson, D.P., Thiesse, M., Dempsey, L.D. and Millar, S.J. (1993). Genomic cloning, physical mapping, and expression of human type 2 cystatin genes. Crit Rev Oral Biol Med 4, 573–580. 10) Dickinson, D.P., Zhao, Y. and Sciliano, K.J. (1994). Direct mapping of seven gene encoding human type 2 cystatins to a single site located at 20p11.2. Genomics 24, 172–175. 11) Freije, J.P., Abrahamson, M., Olafsson, G.I., Velasco, G., Grubb, A. and Lopez-Otin, C. (1991). Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor. J Biol Chem 266, 20538– 20543. 12) Freije, J.P., Pendas, A.M., Velasco, G., Rosa, A., Abrahamson, M. and Lopez-Otin, C. (1993). Localization of the human cystatin D gene (CST5) to chromosome 20p11.21 by in situ hybridization. Cytogenet Cell Genet 62, 29–31. 13) Haga, T. and Minaguchi, K. (1999). Sequence variations of the CST2 gene related to the polymorphism of salivary cystatin SA. J Den Res 78, 835–839. 14) Isemura, S., Saitoh, E. and Sanada, K. (1984). Isolation and amino acid sequence of SAP-1, an acidic protein of human whole saliva, and sequence homology with human gamma-trace. J Biochem 96, 489–498. 15) Isemura, S., Saitoh, E. and Sanada, K. (1986). Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S. FEBS Lett 198, 145–149. 16) Isemura, S., Saitoh, E. and Sanada, K. (1987). Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor. 17). 18). 19). 20). 21). 22). 23). 24). (Cystatin SA) structurally closely related to cystatin S. J Biochem 102, 693–704. Palsdottir, A., Jonsdottir, S., Abrahamson, M., Grubb, A. and Jensson, O. (1990). Three RFLPs at the 3⬘ end of the cystatin C gene, the disease gene in hereditary cystatin C amyloid angiopathy (HCCAA) in Iceland. Nucleic Acids Res 18, 7471. Saitoh, E., Isemura, S., Sanada, K. and Ohnishi, K. (1991). The human cystatin gene family: Cloning of three members and evolutionary relationship between cystatins and BowmanBirk proteinase inhibitors. Biomed Biochem Acta 50, 599–605. Saitoh, E., Isemura, S., Sanada, K. and Ohnishi, K. (1992). Characterization of two members (CST4 and CST5) of the cystatin gene family and molecular evolution of cystatin genes. Agents Actions Suppl 38, 340–348. Saitoh, E., Kim, H.S., Smithies, O. and Maeda, N. (1987). Human cysteine proteinase inhibitors: Nucleotide sequence analysis of three members of the cystatin gene family. Gene 61, 329–338. Saitoh, E., Sabatini, L.M., Eddy, R.L., Shows, T.B., Azen, E.A., Isemura, S. and Sanada, K. (1989). The human cystatin C Gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20. Biochem Biophys Res Commu 162, 1324–1331. Schnittger, S., Gopal Rao, V.V.N., Abrahamson, M. and Hansmann, I. (1993). Cystatin C (CST3), the candidate gene for hereditary cystatin C amyloid angiopathy (HCCAA), and other members of the cystatin gene family are clustered on chromosome 20p11.2. Genomics 16, 50–55. Shintani, M., Minaguchi, K., Isemura, S., Saitoh, E., Sanada, K. and Semba, T. (1994). Genetic polymorphisms of CST2 locus coding for cystatin SA. Hum Genet 94, 45–49. Thiesse, M., Millar, S.J. and Dickinson, D.P. (1994). The human type 2 cystatin gene family consists of eight to nine members, with at least seven genes clustered at a single locus on human chromosome 20. DNA Cell Biol 13, 97–116.. Reprint requests to: Dr. Kiyoshi Minaguchi Department of Forensic Odontology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan.

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Fig. 2 shows a restriction map of type 2 cystatin gene family reported by Saitoh et al
Table 1 Relationship between Sac I RFLP genotypes and salivary cystatin SA (CST2) genotypes Cystatin SA Sac I RFLP genotype (CST2) genotypes 8.3/8.3 8.3/3.5 3.5/3.5 Total

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