Cloning and analysis of the promoter region of Schistosoma japonicum 8k CaBP gene

Abstract

Abstract: Objective To clone and analyze the promoter sequence of Schistosoma japonicum 8k Calcium binding protein(CaBP) (Sj8CaBP) gene. Methods The genomic DNA was extracted from S. japonicum adult worms. The DNA library was constructed according to the user’s manual for Clontech Universal Genome WalkerTM Kit, and the gene-specific primer 1 and 2 (GSP1 and GSP2) of Sj8CaBP gene were designed and synthesized. GSP1 and GSP2 were coupled with the adaptor primer 1 and 2 (AP1 and AP2), then nested PCR was performed. After proper purification, the PCR product was linked to pMD-18 T vector, and the recombinant T-vector was sequenced, analyzed with the promoter prediction software and compared with the cDNA sequence of Sj8CaBP. Results The cloned Sj8CaBP gene was about 1 079 bp in length (GenBank accession No.AY262018), which contained a full open-reading frame with 1 intron and 2 exons. The 5’UTR has a putative promoter region including a TATA-box and a CCAAT-box. The GC-box binding region was not found in this gene, nor was that for active protein 1 (AP-1) often seen in other schistosome genes. Conclusion A 1 079 bp Sj8CaBP gene fragment (GenBank accession No.AY262018) was obtained from a S. japonicum genomic DNA library, containing a promoter region with TATA-box, CCAAT-box, 1 intron and 2 extrons.

CLC Number:

R382.24
Q78

PENG Hong-juan, CHEN Xiao-guang, ZHOU Xiao-hong, SHEN Shu-man. Cloning and analysis of the promoter region of Schistosoma japonicum 8k CaBP gene[J]. Journal of Southern Medical University, 2004, 24(08): 869-872.

References

[1] WHO. Report of the WHO informal consultation on Schistosomiasis control[M]. Geneva, 1998.2-4.
[2] Unnasch T. The filarial genome project[J]. Parasitol Today, 1994,10(3): 415-6
[3] Gloria R, Mark D, Bento S, et al. Identification of new Schistosoma mansoni genes by the EST strategy using a directional cDNA library[J]. Gene, 1995, 152(2): 141-7.
[4] 陈晓光,李华,彭鸿娟.日本血吸虫尾蚴cDNA文库的构建及分析[J].中国寄生虫学与寄生虫病杂志,2002,20(1):39-41.Chen XG, Li H, Peng HJ, et al. Construction and analysis of the Schistosoma japonicum cercariae cDNA library[J]. Zhongguo Jishengchong Xue Yu Jishengchong Bing Zazhi, 2002, 20(1): 39-41.
[5] Peng HJ, Chen XG, Wang XZ, et al. Analysis of the gene expression profile of Schistosoma japonicum cercariae by expressed sequence tag strategy[J]. Parasitol Res, 2003, 90(4): 287-93.
[6] 彭鸿娟,陈晓光,李华,等.几种日本血吸虫新基因表达特性的分析[J].中国人兽共患病杂志,2003,19(2):46-9.Peng HJ, Chen XG, Hua L, et al. Analysis of the expression character for several Schistosomajaponicum novel genes[J]. Zhongguo Renshou Gonghuanbing Zazhi, 2003, 19(2): 46-9.
[7] 许行,彭鸿娟,陈晓光.日本血吸虫Mr为8000钙结合蛋白基因的表达免疫反应性评价[J].第一军医大学学报,2003,23(7):706-8.Xu X, Peng HJ, Chen XG. Expression of Schistosoma japonicum 8kDa calcium-binding protein gene and evaluation of the immunoreactivity of its fusion protein[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2003, 23(7): 706-8.
[8] Wippersteg V, Kapp K, Kunz W, et al. Characterization of the cysteine protease ER60 in transgenic Schistosoma mansoni larvae[J].Int J Parasitol, 2002, 32: 1219-24.
[9] Wippersteg V, Kapp K, Kunz W, et al. HSP70-controlled GFP expression in transiently transformed schistosomes[J]. Mol Biochem Parasitol, 2002, 120: 141-50.
[10] Levy HR, Schechter I.1995. Activity of the promoter of the hsp70gene of the parasitic helminth Schistosoma mansoni in the mammalian CHO cell-line[J]. Biochem Biophys Acta, 1995, 1263: 96-8.
[11] Serra E, Zemzoumi K, Dissous C. Deletion analysis of the Schistosoma mansoni 28-kDa glutathione S-transferase gene pro- moter in mammalian cells--importance of a proximal activator- protein-1 site[J]. Eur J Biochem, 1997, 248: 13-9.
[12] John EH, Martin R. in: John EH. Extraction and purification of nucleic acids from Schistosomes[A]. Protocols in molecular parasitology[M]. Humana Press Inc, 1993.130-3.
[13] 姜泊,张亚历,周殿元,等.冻溶法从琼脂糖凝胶中回收DNA[A].分子生物学常用实验方法[M].北京:人民军医出版社,1996.53.
[14] Lemon B, Tjian R. Orchestrated response: a symphony of transcription factors for gene control[J]. Genes Dev, 2000, 14: 2551-69.
[15] Smale ST. Core promoters: active contributors to combina-torial gene expression[J]. Genes Dev, 2001, 15: 2503-8.
[16] Kutach AK, Kadonaga JT. The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters[J]. Mol Cell Biol, 2000, 20: 4754-64.