Application of Species-specific DNA Probe to Field Samples of Alexandrium tamarense (Lebour) Balech

조은섭,김기영,박형식,김학균,문성기,이재동,Cho, Eun-Seob,Kim, Gi-Young,Park, Hyung-Sik,Kim, Hak-Gyoon,Moon, Sung-Ki,Lee, Jae-Dong Korean Society of Life Science 2002 생명과학회지 Vol.12 No.3

독성이 있는 특이한 편모충인 Alexandrium tamarense의 구별하기 위한 종특이적인 형광 DNA탐침 AT1이 서로 다른 3가지의 고정액에서, 그리고 배양기간의 차이와 whole cell hybridization을 사용하는 광학 현미경 또는 형광 현미경에 의한 세포밀도 측정에 있어서 hiding activity를 비교하는 방법으로 여러 다른 종의 실험에 사용되어졌고, 그의 결과를 보고하는 바이다. 형광 DNA탐침 AT1은 특이적으로, A. tamarense에 결합했지만, 형태학적으로 유사한 다른 편모충에는 결합하지 않았다. 특히 형태적으로 A. tamarense에 유사한 A. catenella는 형광 DNN탐침 AT1에 결합하지 않았다. A. tamarense은 다른 세가지 고정액을 처리하였을 때, 고정액과 상관없이 강한 형광신호를 발산하였다. 추가해서 말하면, 배양기간에 관계없이 형광 DNA탐침 AT1의 biding activity는 강했다. 광학 현미경에 의해서 측정 되어지기 보다는 형광현미경에 의해서 세포밀도가 측정되었다. 형광 DNA탐침 AT1을 이용한 A. tamarense의 동정과 계산은 이 분야에서 새로운 생물독성 감시와 예측 시스템에 기여 할 것이다. Fluorescent species-specific DNA probe (AT1) of toxic dinoflagellate Arexandrium tamarense was tested on several other species, on comparison of binding activity at different preservatives for fixation of the cells, at different culture age and estimation of cell density by light microscope or epifluorescent microscope using whole cell hybridization. Th AT1 probe specifically bound to Alexandrium tamarense, whereas it did not bind to other phytoplankton, in particular Alexandrium catenella, morphologically similar to Alexandrium tamarense, could not react to AT1 probe. When cells were fixed with all three preservatives, labeling cells of Alexandrium tamarense emitted strong fluorescent signal intensity. In addition, regardless culture days, binding activity with AT1 probe was strong. The tell densities estimated by epifluorescent microscope were than those estimated by light microscope. The enumeration and identifying of Arexandriurn tamarense using DNA probe method will be contributed to a new biotoxin monitoring and prediction system in field.

Molecular Analysis of Complete SSU to LSU rDNA Sequence in the Harmful Dinoflagellate Alexandrium tamarense (Korean Isolate, HY970328M)

Ki, Jang-Seu,Han, Myung-Soo The Korean Society of Oceanography 2005 Ocean science journal Vol.40 No.3

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellate Alexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report in Alexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison with Prorocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton, A. tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities of Alexandrium genus, particularly of the tamarensis complex.

Species Identification of Nontoxic Alexandrium tamarense (Dinophyceae) from Chinhae Bay, Korea, Using Molecular Probes

Cho, Eun-Seob Korean Society of Life Science 2004 생명과학회지 Vol.14 No.2

진해만에서 분리한 무독성 Alexandrium tamarense와 이미 보고된 독성을 가진 A. tamarense종 간의 접속공, 제1상판, 후속공의 형태를 상호 비교하였다. 형태적으로 독성종과 거의 일치되지만, HPLC나 생체실험에서 진해만에서 새롭게 분리한 종은 독성이 전혀 나타나지 않았다. lectin반응결과 무독종 A. tamarense은 PNA와 강한 반응을 보여, 세포표면에 lactose나 galactose와 같은 물질이 많이 분포하고 있음을 보였다. 또한, 단백질 전기영동시 무독종은 유독종과 달리 21 kDa 정도 부위에서 종 특이적 밴드를 보였다. 따라서 PNA lectin이나 면역학적 방법을 이용하면 무독종 A. famarense을 신속하게 동정하는데 이용될 수 있다. The morphology of the apical pore complex, the first apical plate and the posterior sulcal plates in a new isolate of Alexandrium tamarense (Lebour) Balech from the Bay of Chinhae was compared with other that of toxic strains of A. tamarense previously isolated from Korean waters. Although this isolate was morphologically identical to these toxic strains, high performance liquid chromatography and mouse bioassay showed no evidence of toxin production. The nontoxic A. tamarense strain showed a strong positive binding activity with PNA lectin, indicating a high density of lactose and galactose residues on the cell surface, and in SDS-PAGE and Western blot analysis a unique protein of about 21-kDa molecular sizes was observed. These findings demonstrate that the use of PNA and immunobioassay could be used to discriminate between toxic and nontoxic strains of A. tamarense.

유독와편모조류 Alexandrium tamarense와 Alexandrium catenella의 성장에 미치는 조도의 영향

권형규 ( Hyeong Kyu Kwon ),박지아 ( Ji A Park ),양한섭 ( Han Soeb Yang ),오석진 ( Seok Jin Oh ) 한국환경과학회 2013 한국환경과학회지 Vol.22 No.12

The effects of irradiance on the growth of toxic dinoflagellates Alexandrium tamarense (Masan Bay strain) and Alexandrium catenella (Jinhae Bay strain) were investigated in the laboratory. At 15℃ and 30 psu for A. tamarense and 25℃ and 30 psu for A. catenella, the irradiance-growth curve showed the maximum growth rate (μmax) of 0.31 day-1 with half-saturation photon flux density (PFD) (KI) of 44.53 μmol m-2 s-1, and a compensation PFD (Ic) was 20.67 μmol m-2 s-1 for A. tamarense, and μmax of 0.38 day-1 with KI of 59.53 μmol m-2 s-1, and Ic was 40.80 μmol m-2 s-1 for A. catenella. The Ic equated to a depth of 8~9 m from March to June for A. tamarense and 6~7 m from March to June for A. catenella. These responses suggested that irradiance at the depth near the middle layer in Masan Bay would provide favorable conditions for two species.

Distribution of Alexandrium tamarense in Drake Passage and the Threat of Harmful Algal Blooms in the Antarctic Ocean

Ho, King-Chung,Kang, Sung-Ho,,Lam Ironside H.Y.,Ho, dgkiss I.John Korea Institute of Ocean ScienceTechnology 2003 Ocean and Polar Research Vol.25 No.4

While phytoplankton diversity and productivity in the Southern Ocean has been widely studied in recent years, most attention has been given to elucidating environmental factors that affect the dynamics of micro-plankton (mainly diatoms) and nano-plankton (mainly Phaeocystis antarctica). Only limited effects have been given to studying the occurrence and the potential risks associated with the blooming of dinoflagellates in the relevant waters. This study focused on the appearance and toxicological characteristics of a toxic dinoflagellate, Alexandrium tamarense, identified and isolated from the Drake Passage in a research cruise from November to December 2001 The appearance of A. tamarense in the Southern Ocean indicates the risk of a paralytic shellfish poisoning (PSP) outbreak there and is therefore of scientific concern. Results showed that while the overall quantity of A. tamarense in water samples from 30meters below the sea surface often comprised less than 0.1% of the total population of phytoplankton, the highest concentration of A. tamarense (20 cells $L^{-1}$) was recorded in the portion of the Southern Ocean between the southern end of South America and the Falkland Islands. Waters near the Polar Front contained the second highest concentrations of 10-15 cells $L^{-1}$. A. tamarense was however rarely found in waters near the southern side of the Polar Front, indicating that cold sea temperatures near the Antarctic ice does not favor the growth of this dinoflagellate. One strain of A. tamarense from this cruise was isolated and cultured for further study in the laboratory. Experiments showed that this strain of A. tamarense has a high tolerance to temperature variations and could survive at temperatures ranging from $5-26^{\circ}C$. This shows the cosmopolitan nature off. tamarense. With regard to the algal toxins produced, this strain of A. tamarense produced mainly C-2 toxins but very little saxitoxin and gonyailtoxin. The toxicological property of this A. tamarense strain coincided with a massive death of penguins in the Falkland Islands in December 2002 to January 2003.

A Molecular Phylogenetic Study on Korean Alexandrium catenella and A. tamarense Isolates (Dinophyceae) Based on the Partial LSD rDNA Sequence Data

Kim, Keun-Yong,Kim, Chang-Hoon The Korean Society of Oceanography 2004 Journal of the Korean Society of Oceanography Vol.39 No.3

Sequences of the large subunit ribosomal (LSD) rDNA D1-D2 region of Alexandrium catenella(=A. sp. cf. catenella) and A. tamarense isolates, which were collected along the Korea coasts, were analyzed to understand their phylogenetic relationships and geographical distributions. All A. catenella and A. tamarense isolates belonged to the A. tamarense/catenella/fundyense complex and were grouped with the North American and temperate Asian ribotypes, respectively, regardless of the presence or absence of a ventral pore in the first apical plate. A consistent and peculiar characteristic that differentiated the Alexandrium isolates was amplification of a second PCR product with a lower molecular weight in addition to the predicted one; ten A. catenella isolates belonging to the temperate Asian ribotype yielded this additional PCR product. Sequence alignment revealed that the shorter PCR product resulted from an unusual large deletion of 87 bp in the LSD rDNA D1 domain. The North American and temperate Asian ribotypes were prevalent along the Korean coasts without geographical separation. Given the high genetic homogeneity among widely distributed Alexandrium populations, each ribotype appeared to be pandemic rather than to constitute a distinct regional population.

독성 Alexandrium tamarense 의 EST 분석 및 삭시톡신 생합성 유전자의 확인

장만(Chang, Man),이주연(Lee, Ju-Yeon),정영재(Chung, Young-Jae),이건섭(Lee, Gun-Sup),김동균(Kim, Dong-Giun),이택견(Lee, Taek-Kyun) 한국산학기술학회 2013 한국산학기술학회논문지 Vol.14 No.7

A. tamarense로부터 ESTs library를 제작하였다. 이들의 염기서열을 분석하여 STX 생합성 관련 유전자를 클로닝하 였다. 연구결과 827 클론의 염기서열이 분석되었고, 564개의 EST가 GenBank에서의 Blast search를 사용하여 기능에 따라 분류되었다. EST에서의 주요 유전자는 cellular organization, cell metabolism, energy, cell cycle과 DNA processing, cellular transport와 transport, cell rescue, defense, death와 aging 및 transcription 등으로 분석되었다. 특히 S-adenosylmethionine synthetase와 H2A histone family 유전자의 발현이 독성 A. tamarense에서 증가하였다. 이러한 결과는 두 개의 유전자가 A. tamarense에서의 삭시톡신 생합성을 검출하기 위한 좋은 바이오마커가 될 수 있음을 보여준다. Expressed sequence tag (EST) library was constructed from A. tamarense. Base sequences of EST clones were analyzed and saxitoxin biosynthesis-related genes were cloned. Sequences of 827 clones were analyzed and 564 EST were functionally clustered using Blast searches against GenBank. Main genes in the EST had functions on cellular organization, cell metabolism, energy, cell cycle and DNA processing, cellular transport and transport, cell rescue, defense, death and aging, and transcription. Moreover, expression of S-adenosylmethionine synthetase and H2A histone family genes were increased in the toxic A. tamarense. These results show that two genes could be a good biomarkers for the detection of saxitoxin biosynthesis in the A. tamarense.

Phylogenetic position of five Korean strains of Alexandrium tamarense(Dinophyceae), based on internal transcribed spacers ITS1 and ITS2 including nuclear-encoded 5.85 rRNA gene sequences

조은섭,이삼근,김익수,Cho, Eun-Seob,Lee, Sam-Geun,Kim, Ik-Soo Korean Society of Life Science 2002 생명과학회지 Vol.12 No.6

알렉산드륨 적조생물의 리보소옴 알엔에이 유전자의 ITS1, 2 및 5.8S부위를 대상으로 종간 혹은 종내의 유전적 다양도를 조사하기 위하여 지리적으로 격리된 33 스트레인 유전자의 염기서열를 비교했다. 진해만에서 분리된 AT-2, AT-6, AT-10, AT-A, AT-B 5클론은 일본종 OFX151-A과 동일한 유전자임을 발견했다. ITS부위에서 가장 짧은 종은 A. margalefi로 481 bp이며 가장 긴 종은 A. affine으로 528 bp로 나타났다. ITS1과 ITS2 염기서열에 대한 상호관계는 역으로 나타낸 반면에, G+C 함량에 대한 상호관계는 플러스로 나타났다. 유전적 변이율은 0.3% (1 bp)에서 53% (305 bp)였다. A. tamarense과 가장 적게 유전적 변이율을 보인 종은 A. fundyense(1.2-2.3% = 6-12 bp)인 반면에, A. catenella와는 큰 변이율 (19.8% = 102 bp)을 보였고, A. catenella와 A. fundyense은 19.7% 상이하였다. 알렉산드륨 적조생물의 bootstrap은 약하게 지지되는 데도 불구하고, A. catenella 분리종은 독립적인 그룹으로 형성하여 상호간에는 강력한 bootstrap 값은 PAUP과 NJ 분석에서 보였다. A. cohorticula와 A. frateculus 적조생물은 항상 sub-group 내에서 높은 bootstrap을 가졌다. 결론적으로 ITS부위의 염기서열 분석은 알렉산드륨 적조생물의 집단내 혹은 집단간의 계통분류을 밝히는데 유용한 것으로 보였다. In order to measure the inter- and intraspecific genetic divergences within the genus Alexandrium, the variations within the internal transcribed spacer (ITS1 and ITS2) regions and 5.85 ribosomal RNA gene of eight Alexandrium species were examined for 33 strains from diverse geographical locations by direct sequencing. Five isolates of A. tamarense (AT-2, AT-6, AT-10, AT-A and AT-B) from Jinhae Bay, Korea were found to be completely identical to a Japanese strain OFX151-A. The length of the amplified ITSI-5.85-ITS2 region varied from 481 nucleotides (in A. margalefi) to 528 nucleotides (in A. affine CU1-1). ITS1 and ITS2 nucleotide lengths were negatively correlated, whereas a positive correlation was found between their G+C content. The degree of sequence divergence ranged from 0.3% (1 bp) to a maximum of 53% (305 Up). Pairwise sequence comparisons revealed a small degree of divergence between A. tamarense and A. Pundyense isolates (1.2 - 2.3% = 6-12 bp), but a high degree of divergence between A. tamarense and A. catenella (19.8% = 102 bp), and between A. catenella and A. Pundyense (19.7%). Although most nodes were weakly supported by bootstrap values, some types tend to form independent molecular groups. A. catenella isolates also formed an independent molecular sub-group, with relaticula strong bootstrap values (94% or 85% and 79% or 98%, respectively in PAUP and NJ trees). Interestingly, A. cohorticula and A. frateculus always clustered within the same sub-group, this result being supported by strong bootstrap values. Our results indicate that the ITS regions provide useful informations on hierarchical population genetic structure and a high phylogenetic resolution in intraspecific and interspecific Alexandrium population.

The First Appearance of Toxic Dinoflagellate Alexandrium tamarense (Gonyaulacales, Dinophyceae) Responsible for the PSP Contaminations in Gamak Bay, Korea

Shin, Hyeon-Ho,Yoon, Yang-Ho,Kawami, Hisae,Iwataki, Mitsunori,Matsuoka, Kazumi The Korean Society of Phycology 2008 ALGAE Vol.23 No.4

In Gamak Bay, Paralytic Shellfish Poisoning (PSP) was first detected from seafoods in 2003, however the toxin source is unknown yet. In this study, we report potential PSP producers of toxic dinoflagellates, describing morphology and abundance of cysts isolated from surface sediment of Gamak Bay. The most abundant type in these cysts was characterized with ellipsoidal and transparent wall identical to Alexandrium catenella and/or A. tamarense. Germination experiment of the cysts revealed that all motile cells germinated were morphologically identified as A. tamarense. This result suggests that A. tamarense may relate to PSP contaminations in Gamak Bay. Moreover, bottom water temperature in Gamak Bay is favorable for germination of A. tamarense cysts. Further studies are required to carry out the PSP monitoring for preventing the risk of PSP events that may outbreak in future at Gamak Bay.

The First Appearance of Toxic Dinoflagellate Alexandrium tamarense (Gonyaulacales, Dinophyceae) Responsible for the PSP Contaminations in Gamak Bay, Korea [Note]

신현호,윤양호,Hisae Kawami,Mitsunori Iwataki,Kazumi Matsuoka 한국조류학회I 2008 ALGAE Vol.23 No.4

In Gamak Bay, Paralytic Shellfish Poisoning (PSP) was first detected from seafoods in 2003, however the toxin source is unknown yet. In this study, we report potential PSP producers of toxic dinoflagellates, describing morphology and abundance of cysts isolated from surface sediment of Gamak Bay. The most abundant type in these cysts was characterized with ellipsoidal and transparent wall identical to Alexandrium catenella and/or A. tamarense. Germination experiment of the cysts revealed that all motile cells germinated were morphologically identified as A. tamarense. This result suggests that A. tamarense may relate to PSP contaminations in Gamak Bay. Moreover, bottom water temperature in Gamak Bay is favorable for germination of A. tamarense cysts. Further studies are required to carry out the PSP monitoring for preventing the risk of PSP events that may outbreak in future at Gamak Bay.