Abstract
The mortality of sea squirts, Halocynthia roretzi, with softness syndrome threatens the sea squirt aquaculture industry in Asian countries. The molecular approach to understanding the pathogenesis of softness syndrome began with differential gene expression analysis of tissues from normal and dying organisms. In the present study, we show that the expression of Halocynthia roretzi metalloproteinase (HrMMP) was significantly upregulated in the tissues of dying organisms through screening of differentially expressed genes, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR. HrMMP is composed of 482 amino acids, contains a conserved domain found in the astacin family, and has typical metalloproteinase activity. To discriminate between the differential expression of the HrMMP gene in normal and dying organisms, we cloned the HrMMP gene promoter and identified a polymorphism in the HrMMP promoter region that resulted in distinct polymorphisms (G/T) at position - 308 bp. These results suggest that organisms with the GT genotype may have more resistance to softness syndrome than those with the TT genotype. These findings suggest that the HrMMP promoter polymorphism may be associated with an increased risk of softness syndrome in cultivated sea squirts and should be evaluated as a candidate molecular marker for the selective breeding of softness syndrome-resistant sea squirts.
Similar content being viewed by others
References
Ailenberg M, Silverman M (2000) Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS). Biotechniques 29:1018–1020
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Bond JS, Beynon RJ (1995) The astacin family of metalloendopeptidases. Protein Sci 4:1247–1261
Breathnach R, Chambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383
Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S (1997) The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res 25:3235–3241
Chen SL, Li J, Deng SP, Tian YS, Wang QY, Zhuang ZM, Sha ZX, Xu JY (2007) Isolation of female-specific AFLP markers and molecular identification of genetic sex in half-smooth tongue sole (Cynoglossus semilaevis). Mar Biotechno l 9:273–280
Corden J, Wasylyk B, Buchwalder A, Sassone-Corsi P, Kedinger C, Chambon P (1980) Promoter sequences of eukaryotic protein-coding genes. Science 209:1406–1414
Dumermuth E, Eldering JA, Grünberg J, Jiang W, Sterchi EE (1993) Cloning of the PABA peptide hydrolase alpha subunit (PPH alpha) from human small intestine and its expression in COS-1 cells. FEBS Lett 335:367–375
Elaroussi MA, DeLuca HF (1994) A new member to the astacin family of metalloendopeptidases: a novel 1,25-dihydroxyvitamin D-3-stimulated mRNA from chorioallantoic membrane of quail. Biochim Biophys Acta 1217:1–8
Fang S, Jin X, Wang R, Li Y, Guo W, Wang N, Wang Y, Wen D, Wei L, Zhang J (2005) Polymorphisms in the MMP1 and MMP3 promoter and non-small cell lung carcinoma in North China. Carcinogenesis 26:481–486
Fukagawa M, Suzuki N, Hogan BL, Jones CM (1994) Embryonic expression of mouse bone morphogenetic protein-1 (BMP-1), which is related to the Drosophila dorsoventral gene tolloid and encodes a putative astacin metalloendopeptidase. Dev Biol 163:175–183
Gorbea CM, Marchand P, Jiang W, Copeland NG, Gilbert DJ, Jenkins NA, Bond JS (1993) Cloning, expression, and chromosomal localization of the mouse meprin beta subunit. J Biol Chem 268:21035–21043
He H, Linder DP, Rodgers KR, Chakraborty I, Arif AM (2004) A thiazole-containing tripodal ligand: synthesis, characterization, and interactions with metal ions and matrix metalloproteinases. Inorg Chem 43:2392–2401
Hultmark D, Klemenz R, Gehring WJ (1986) Translational and transcriptional control elements in the untranslated leader of the heat-shock gene hsp22. Cell 44:429–438
Hwang IT, Kim YJ, Kim SH, Kwak CI, Gu YY, Chun JY (2003) Annealing control primer system for improving specificity of PCR amplification. Biotechniques 35:1180–1184
Jansen RC, Nap JP (2004) Regulating gene expression: surprises still in store. Trends Genet 20:223–225
Kadonaga JT (2002) The DPE, a core promoter element for transcription by RNA polymerase II. Exp Mol Med 34:259–264
Katagiri C, Maeda R, Yamashika C, Mita K, Sargent TD, Yasumasu S (1997) Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells. Int J Dev Biol 41:19–25
Lara-Pezzi E, Gómez-Gaviro MV, Gálvez BG, Mira E, Iñiguez MA, Fresno M, Martínez-A C, Arroyo AG, López-Cabrera M (2002) The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression. J Clin Invest 110:1831–1838
Martoglio B, Dobberstein B (1998) Signal sequences: more than just greasy peptides. Trends Cell Biol 8:410–415
McPherson MJ, Moller SJ (2000) PCR the basics from background to bench. BIOS Scientific Publishers, Oxford
Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6
Okamoto T, Akuta T, Tamura F, Der Vliet A, Akaike T (2004) Molecular mechanism for activation and regulation of matrix metalloproteinases during bacterial infections and respiratory inflammation. Biol Chem 385:997–1006
Park CH, Lee MJ, Ahn J, Kim S, Kim HH, Kim KH, Eun HC, Chung JH (2004) Heat shock-induced matrix metalloproteinase (MMP)-1 and MMP-3 are mediated through ERK and JNK activation and via an autocrine interleukin-6 loop. J Invest Dermatol 123:1012–1019
Passmore AJ, Jarman SN, Swadling KM, Kawaguchi S, McMinn A, Nicol S (2006) DNA as a dietary biomarker in Antarctic krill, Euphausia superba. Mar Biotechnol 8:686–696
Pearce E, Tregouet DA, Samnegard A, Morgan AR, Cox C, Hamsten A, Eriksson P, Ye S (2005) Haplotype effect of the matrix metalloproteinase-1 gene on risk of myocardial infarction. Circ Res 97:958–960
Saiki RK, Walsh PS, Levenson CH, Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA 86:6230–6234
Shimell MJ, Ferguson EL, Childs SR, O’Connor MB (1991) The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1. Cell 67:469–481
Sugimoto M, Yoshida S, Kennedy S, Deguchi M, Ohara N, Maruo T (2006) Matrix metalloproteinase-1 and -9 promoter polymorphisms and endometrial carcinoma risk in a Japanese population. J Soc Gynecol Investig 13:523–529
Takahara K, Lyons GE, Greenspan DS (1994) Bone morphogenetic protein-1 and a mammalian tolloid homologue (mTld) are encoded by alternatively spliced transcripts which are differentially expressed in some tissues. J Biol Chem 269:32572–32578
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Titani K, Torff HJ, Hormel S, Kumar S, Walsh KA, Rodl J, Neurath H, Zwilling R (1987) Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis. Biochemistry 26:222–226
Woo S, Yum S, Jung JH, Shim WJ, Lee CH, Lee TK (2006) Heavy metal-induced differential gene expression of metallothionein in Javanese medaka, Oryzias javanicus. Mar Biotechnol 8:654–662
Wozney JM, Rosen V, Celeste AL, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA (1988) Novel regulators of bone formation: molecular clones and activities. Science 242:1528–1534
Yasumasu S, Iuchi I, Yamagami K (1994) cDNA and the genes of HCE and lCE, fwo constituents of the medaka hatching enzyme. Dev Growth Differ 36:241–250
Acknowledgment
This study was supported by Multi-Component Project (Sea Squirt Aquaculture, RP-2008-BT-001) of the National Fisheries Research and Development Institute in Korea and by fisheries research and development and development funds granted the Korean Ministry of Maritime affairs and Fisheries (2007).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the linked to the electronic supplementary material.
ESM Table 1
Primer sequences used in cDNA synthesis and ACP-based GeneFishing PCR (GIF 19 KB)
Rights and permissions
About this article
Cite this article
Cho, H.K., Nam, BH., Kong, H.J. et al. Identification of Softness Syndrome-Associated Candidate Genes and DNA Sequence Variation in the Sea Squirt, Halocynthia roretzi . Mar Biotechnol 10, 447–456 (2008). https://doi.org/10.1007/s10126-008-9084-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10126-008-9084-y