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( Sirawut Klinbunga ),( Piti Amparyup ),( Rungnapa Leelatanawit ),( Anchalee Tassanakajon ),( Ikuo Hirono ),( Takashi Aoki ),( Padermsak Jarayabhand ),( Piamsak Menasveta ) 생화학분자생물학회 2004 BMB Reports Vol.37 No.2
A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Halwtis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N= 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CHHO3) and H. varia(CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N= 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.
Klinbunga, Sirawut,Amparyup, Piti,Leelatanawit, Rungnapa,Tassanakajon, Anchalee,Hirono, Ikuo,Aoki, Takashi,Jarayabhand, Padermsak,Menasveta, Piamsak Korean Society for Biochemistry and Molecular Biol 2004 Journal of biochemistry and molecular biology Vol.37 No.2
A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.
Imjongjirak, Chanprapa,Klinbunga, Sirawut,Sittipraneed, Siriporn Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.1
Major Royal Jelly Protein cDNAs of Apis cerana (AcMRJP) were cloned and characterized. The open reading frames (ORFs) of the AcMRJP1 and AcMRJP2 genes were 1302 and 1392 nucleotides, encoding 433 and 463 amino acid residues, respectively. The sequence divergences between AcMRJP1 and AcMRJP2 and their corresponding protein families in A. mellifera were 0.0618 and 0.0934 at the nucleotide level and 0.0912 and 0.1438 at the protein level, respectively. Phylogenetic analysis supports the orthologous similarity between these proteins. The deduced amino acids indicated high essential amino acid contents of AcMRJP1 and AcMRJP2 (47.5 and 44.8%, respectively). The genomic organization of both AcMRJP1 and AcMRJP2 was determined. Both the AcMRJP1 (3663 bp) and AcMRJP2 (3963 bp) genes contained six exons and five introns, where all boundaries conformed to the GT/AG rule. AcMRJP1 and AcMRJP2 cDNAs were cloned into pET17b, and both the recombinant (r) AcMRJP1 (47.9 kDa) and rAcMRJP2 (51.7 kDa) were expressed in the insoluble form. Western blot analysis and N-terminal sequencing of the solubilized proteins revealed successful expression of rAcMRJP1 and rAcMRJP2 in vitro. The yields of the purified rAcMRJP1 and rAcMRJP2 were approximately 20 and 8mg protein per liter of the flask culture, respectively.
( Chanprapa Imjongjirak ),( Sirawut Klinbunga ),( Siriporn Sittipraneed ) 생화학분자생물학회 2005 BMB Reports Vol.38 No.1
Major Royal Jelly Protein cDNAs of Apis cerana (AcMRJP) were cloned and characterized. The open reading frames (ORFs) of the AcMRJP1 and AcMRJP2 genes were 1302 and 1392 nucleotides, encoding 433 and 463 amino acid residues, respectively. The sequence divergences between AcMRJP1 and AcMRJP2 and their corresponding protein families in A. mellifera were 0.0618 and 0.0934 at the nucleotide level and 0.0912 and 0.1438 at the protein level, respectively. Phylogenetic analysis supports the orthologous similarity between these proteins. The deduced amino acids indicated high essential amino acid contents of AcMRJP1 and AcMRJP2 (47.5 and 44.8%, respectively). The genomic organization of both AcMRJP1 and AcMRJP2 was determined. Both the AcMRJP1 (3663 bp) and AcMRJP2 (3963 bp) genes contained six exons and five introns, where all boundaries conformed to the GT/AG rule. AcMRJP1 and AcMRJP2 cDNAs were cloned into pET17b, and both the recombinant (r) AcMRJP1 (47.9 kDa) and rAcMRJP2 (51.7 kDa) were expressed in the insoluble form. Western blot analysis and N-terminal sequencing of the solubilized proteins revealed successful expression of rAcMRJP1 and rAcMRJP2 in vitro. The yields of the purified rAcMRJP1 and rAcMRJP2 were approximately 20 and 8 mg protein per liter of the flask culture, respectively.
Srisuparbh, Duangporn,Klinbunga, Sirawut,Wongsiri, Siriwat,Sittipraneed, Siriporn Korean Society for Biochemistry and Molecular Biol 2003 Journal of biochemistry and molecular biology Vol.36 No.6
An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts >500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of $\alpha$-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.
( Duangporn Srisuparbh ),( Sirawut Klinbunga ),( Siriwat Wongsiri ),( Siriporn Sittipraneed ) 생화학분자생물학회 2003 BMB Reports Vol.36 No.6
An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts >500 bp, were randomly selected and unidirectional sequenced. Fprty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1sequence showed 93 % and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of α-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks Al, A2, B1, and C1 were 115, 55, 50, and 300kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 :A2 :B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJPl of A. mellifera.
Species Identification of Five Penaeid Shrimps Using PCR-RFLP and SSCP Analyses of 16S Ribosomal DNA
Khamnamtong, Bavornlak,Klinbunga, Sirawut,Menasveta, Piamsak Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.4
DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCR-RFLP of 16S $rDNA_{560}$ whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S $rDNA_{560}$. Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S $rDNA_{560}$ of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S $rDNA_{312}$ was as effective as that of 16S $rDNA_{560}$. Differentiation of all shrimp species were successfully carried out by SSCP analysis.