GORO YOSHIZAKI
Department of Marine Biosciences, Tokyo University of Marine Science and Technology

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Cloning and Expression Analysis of a Giant Gourami Vasa-Like cDNA ALIMUDDIN, .; ANDRIYANI, IRMA; JUNIOR, MUHAMMAD ZAIRIN; ARFAH, HARTON; OCTAVERA, ANNA; CARMAN, ODANG; YOSHIZAKI, GORO
HAYATI Journal of Biosciences Vol 18, No 3 (2011): September 2011
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.18.3.135

Abstract

Molecular marker is useful in the development of testicular cells transplantation for detecting donor-derived germ cells in the recipient gonad. In this study, a giant gourami (Osphronemus goramy) vasa-like gene (GgVLG) was cloned and characterized for use as a molecular marker for germ cells in this species. Nucleotide sequence analysis revealed that GgVLG comprises 2,340 bps with an open reading frame of 1,962 bps encoding 653 amino acids. The deduced amino acid sequence contained 17 arginine-glycine or arginine-glycine-glycine motifs and eight conserved motifs belonging to the DEAD-box protein family. The GgVLG sequence showed high similarity to Drosophila vasa, common carp vasa homolog and tilapia vasa homolog for 66.2, 85.9, and 90.7%, respectively. In adult tissues, the GgVLG transcripts were specifically detected in ovary and testis. In situ hybridization analysis showed that GgVLG mRNA was detected in oocytes of the ovary and spermatogonia of the testis. There was no signal detected in the spermatocytes, spermatids and other gonadal somatic cells. Thus, consensus sequences, specific localization of GgVLG mRNA in the germ cells, amino acid sequence similarity and phylogenic analysis all suggest that GgVLG is the giant gourami vasa-like gene. Further, GgVLG can be used as a molecular marker for giant gourami germ cells.
OVER-EXPRESSION OF GENE ENCODING FATTY ACID METABOLIC ENZYMES IN FISH Alimuddin, Alimuddin; Yoshizaki, Goro; Takeuchi, Toshio; Carman, Odang
Indonesian Aquaculture Journal Vol 3, No 2 (2008): (December 2008)
Publisher : Pusat Penelitian dan Pengembangan Perikanan, Badan Penelitian dan Pengembangan Kelautan da

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15578/iaj.3.2.2008.89-106

Abstract

Eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have important nutritional benefits in humans. EPA and DHA are mainly derived from fish, but the decline in the stocks of major marine capture fishes could result in these fatty acids being consumed less. Farmed fish could serve as promising sources of EPA and DHA, but they need these fatty acids in their diets. Generation of fish strains that are capable of synthesizing enough amounts of EPA/DHA from the conversion of α-linolenic acid (LNA, 18:3n-3) rich oils can supply a new EPA/DHA source. This may be achieved by over-expression of genes encoding enzymes involved in HUFA biosynthesis. In aquaculture, the successful of this technique would open the possibility to reduce the enrichment of live food with fish oils for marine fish larvae, and to completely substitute fish oils with plant oils without reducing the quality of flesh in terms of EPA and DHA contents. Here, three genes, i.e. Δ6-desaturase-like (OmΔ6FAD), Δ5-desaturase-like (OmΔ5FAD) and elongase-like (MELO) encoding EPA/DHA metabolic enzymes derived from masu salmon (Oncorhynchus masou) were individually transferred into zebrafish (Danio rerio) as a model to increase its ability for synthesizing EPA and DHA. Fatty acid analysis showed that EPA content in whole body of the second transgenic fish generation over-expressing OmΔ6FAD gene was 1.4 fold and that of DHA was 2.1 fold higher (P<0.05) than those in non-transgenic fish. The EPA content in whole body of transgenic fish over-expressing OmΔ5FAD gene was 1.21-fold, and that of DHA was 1.24-fold higher (P<0.05) than those in nontransgenic fish. The same patterns were obtained in transgenic fish over-expressing MELO gene. EPA content was increased by 1.30-fold and DHA content by 1.33-fold higher (P<0.05) than those in non-transgenic fish. The results of studies demonstrated that fatty acid content of fish can be enhanced by over-expressing gene encoding enzymes involved in fatty acid biosynthesis, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food.
Molecular marker is useful in the development of testicular cells transplantation for detecting donor-derived germ cells in the recipient gonad. In this study, a giant gourami (Osphronemus goramy) vasa-like gene (GgVLG) was cloned and characterized for use as a molecular marker for germ cells in this species. Nucleotide sequence analysis revealed that GgVLG comprises 2,340 bps with an open reading frame of 1,962 bps encoding 653 amino acids. The deduced amino acid sequence contained 17 arginine- ALIMUDDIN, .; ANDRIYANI, IRMA; JUNIOR, MUHAMMAD ZAIRIN; ARFAH, HARTON; OCTAVERA, ANNA; CARMAN, ODANG; YOSHIZAKI, GORO
HAYATI Journal of Biosciences Vol 18, No 3 (2011): September 2011
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.18.3.135

Abstract

Molecular marker is useful in the development of testicular cells transplantation for detecting donor-derived germ cells in the recipient gonad. In this study, a giant gourami (Osphronemus goramy) vasa-like gene (GgVLG) was cloned and characterized for use as a molecular marker for germ cells in this species. Nucleotide sequence analysis revealed that GgVLG comprises 2,340 bps with an open reading frame of 1,962 bps encoding 653 amino acids. The deduced amino acid sequence contained 17 arginine-glycine or arginine-glycine-glycine motifs and eight conserved motifs belonging to the DEAD-box protein family. The GgVLG sequence showed high similarity to Drosophila vasa, common carp vasa homolog and tilapia vasa homolog for 66.2, 85.9, and 90.7%, respectively. In adult tissues, the GgVLG transcripts were specifically detected in ovary and testis. In situ hybridization analysis showed that GgVLG mRNA was detected in oocytes of the ovary and spermatogonia of the testis. There was no signal detected in the spermatocytes, spermatids and other gonadal somatic cells. Thus, consensus sequences, specific localization of GgVLG mRNA in the germ cells, amino acid sequence similarity and phylogenic analysis all suggest that GgVLG is the giant gourami vasa-like gene. Further, GgVLG can be used as a molecular marker for giant gourami germ cells.
Growth, Survival, and Body Composition of Transgenic Common Carp Cyprinus carpio 3rd Generation Expressing Tilapia Growth Hormone cDNA Kurdianto, .; Alimuddin, .; Faridah, Nurly; Yoshizaki, Goro; Nuryati, Sri; Setiawati, Mia
HAYATI Journal of Biosciences Vol 23, No 3 (2016): July 2016
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.23.3.150

Abstract

Transgenic has been known as one of the applicable methods to improve growth performance of cultured fish. This study was performed to evaluate the growth performance, survival, and body composition of the 3rd generation of growth hormone (GH) transgenic common carp (TG). Juveniles (BW: 1.53 ± 0.03 g) were reared for 60 days in 250-L glass aquarium with stocking density of 25 fishes/aquarium. Fishes were fed with commercial feed (protein content 36%), three times a day to satiation. Growth and survival were measured every 20 days. Our results showed that TG fish has 1.49 times higher in average weight growth (p < 0.05) compared with the non-transgenic common carp (NT). Higher total feed consumption, survival, body protein content, protein and lipid retention, hepatosomatic index, and lower feed conversion ratio were also shown on TG fish compared with NT fish (p < 0.05). However, body lipid content and blood glucose level of TG fish were lower (p < 0.05) compared with the NT fish. Total ammonium nitrogen level in rearing media of TG fish was 51.78% lower (p < 0.05) than that of the NT fish. In conclusion, culturing of GH-TG common carp showed potential to achieve high productivity, efficient, and environmental-friendly aquaculture.
ISOLASI DAN KARAKTERISASI PROMOTER β-ACTIN DARI IKAN KERAPU BEBEK (Cromileptes altivelis) Alimuddin, Alimuddin; Nugrahani, Wedaraningtyas; Aliah, Ratu Siti; Sumantadinata, Komar; Faizal, Irvan; Carman, Odang; Yoshizaki, Goro
Jurnal Riset Akuakultur Vol 2, No 2 (2007): (Agustus 2007)
Publisher : Pusat Riset Perikanan, Badan Riset dan Sumber Daya Manusia Kelautan dan Perikanan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15578/jra.2.2.2007.199-210

Abstract

Promoter sebagai regulator ekspresi gen merupakan salah satu faktor yang mempengaruhi keberhasilan transgenesis.  Penelitian isolasi dan karakterisasi promoter ?-actin (ktBA) dari ikan kerapu bebek (Cromileptes altivelis) dalam rangka pembuatan ikan kerapu autotransgenik telah dilakukan. Promoter ?-actin memiliki aktivitas tinggi pada jaringan otot. Sekuens promoter ktBA diisolasi menggunakan metode degenerate PCR. Sekuensing dilakukan menggunakan mesin ABI PRISM 3100. Analisis sekuens menggunakan software BLAST, GENETYX versi 7 dan TFBind. Fragment DNA hasil amplifikasi PCR yang dipotong dari vektor kloning selanjutnya diligasi dengan pEGFPN1 untuk membuat konstruksi pktBA-EGFP. Konstruksi pktBA-EGFP dimikroinjeksi ke embrio ikan zebra (Danio rerio) fase 1 sel untuk menguji aktivitas promoter ktBA. Ekspresi gen EGFP diamati menggunakan mikroskop fluoresens. Analisis sekuens menunjukkan bahwa panjang fragmen DNA hasil amplifikasi PCR sekitar 1,6 kb dan memiliki faktor transkripsi yang conserved pada promoter ?-actin, yaitu CCAAT, CArG dan boks TATA. Selanjutnya, sekuens ktBA dalam konstruksi pktBA-EGFP mampu mengendalikan ekspresi gen EGFP pada jaringan otot embrio ikan zebra yang dimikroinjeksi dengan konstruksi tersebut. Dengan demikian dapat disimpulkan bahwa fragmen DNA hasil amplifikasi PCR tersebut merupakan sekuens promoter ?-actin ikan kerapu bebek. Pembuatan ikan kerapu autotransgenik selanjutnya dapat dilakukan dengan mengganti gen EGFP pada pktBA-EGFP dengan gen-gen asal ikan kerapu yang mengkodekan karakter penting dalam budi daya ikan.Promoter as gene expression regulator is one of the factors affecting the successful of transgenesis. Isolation and characterization of ? -actin promoter (ktBA) from humpback grouper (Cromileptes altivelis) towards generation of autotransgenic grouper have been conducted.  ? -actin promoter has high activity in muscle. Sequence of ktBA promoter was isolated by using degenerate PCR method. Sequencing was performed using ABI PRISM 3100 machine. Analysis of sequences was conducted using BLAST, GENETYX version 7 and TFBind softwares. DNA fragment of PCR amplification product digested from the vector cloning was then ligated with pEGFPN1 to generate pktBA-GFP construct. The construct was microinjected into one-cell stage of zebrafish (Danio rerio) embryos to test the ktBA promoter activity. EGFP gene expression was observed by fluorescence microscope. The result of sequence analysis showed that the length of DNA fragment obtained is about 1.6 kb and containing the evolutionary conserved sequences of transcription factor for ? -actin promoter including CCAAT, CArG and TATA boxes. Furthermore, ktBA sequence in pktBA-EGFP construct could drove GFP expression in muscle of zebrafish embryos injected with the construct. The results suggested that PCR amplification product is the regulator sequence of humpback grouper ? -actin gene. Autotransgenic grouper can be then produced by changing GFP gene fragment of pktBA-EGFP construct with genes from grouper encoding important traits in aquaculture.