Diversity and distribution of free-living benthic heterotrophic flagellates in Botany Bay, Australia

이원제 School of Biological Sciences, University of Sydne 2001 해외박사

This thesis seeks to improve our understanding of the diversity and distribution of free-living benthic heterotrophic flagellates in marine sediments. It aims to document the diversity of heterotrophic flagellates in Australian marine sediments, to estimate their abundance and biomass, to explore factors controlling their abundance and distribution, and to explore the diversity and geographic distribution of heterotrophic flagellates. The diversity of benthic heterotrophic flagellates was examined at various sites around Australia from February 1997 to February 2000. A total of 166 species and 65 genera were found and are described using light microscopy. Although the cumulative curves of species recorded against time suggest that most species present were found, new species were encountered at a slow rate, and about 44 new taxa are reported here although not named for lack of information. This suggests that there are some more undescribed species. The taxonomy, morphological variation, and other aspects of known taxa or poorly known taxa are discussed in detail. The abundance and biomass of heterotrophic flagellates, algae, bacteria, ciliates, dinoflagellates, forminifera and nematodes were estimated monthly at Botany Bay during February 1999 - February 2000. The annual abundance of the heterotrophic flagellates in Botany Bay was in the ranges of 0.46 - 4.70x105 cells/㎤. The majority of heterotrophic flagellates (93 - 100 %) were less than l0μm in length and few flagellates were larger than l0μm. Of the total microbial carbon biomass, heterotrophic flagellates made up about 5 % (but at times up to 35 %) and the contribution of heterotrophic flagellates to the microbial carbon biomass varied monthly among the sites. Heterotrophic flagellates were negatively correlated with grain size and positively correlated with the abundance of bacteria, algae, nematodes and their probable grazers. Using the grazing rates on bacteria previously reported, it can be calculated that heterotrophic flagellates would consume a maximum of 64 % of bacterial standing stock daily in Botany Bay, suggesting an importance of heterotrophic flagellates as bacterivores. This study suggests that in Botany Bay the bacterial abundance to be a potentially important factor for determining the abundance of heterotrophic flagellates, and grain size to be a factor determining their abundance and distribution. The importance of heterotrophic flagellates might vary monthly and among the sites (or habitats). About 700 nominal species and 214 genera of free-living heterotrophic flagellates were produced from a survey of the literature on the distribution of flagellates from marine zones - (excluding dinoflagellates and haptophytes). This survey reveals that more than half of the species has been reported from a single location, generally unusual or poorly studied habitats. This implies that heterotrophic flagellates are often endemic. In a series of original surveys of flagellates, about 34 % were reported from a single location - again suggestive of endemism. When the communities from these surveys are compared using the clustering algorithm in PRIMER, there is - in contrast - no evidence of endemism because the communities from geographic regions do not cluster together. Communities cluster on the type of habitat from which they were drawn and this suggests that geographic location may play no part in the makeup of communities. The conflict of insights from information on species and information on communities creates uncertainty over the geographical distribution of flagellates. This is probably because the actual distribution of these organisms is obscured by factors extrinsic to their distribution - principally issues relating to under-reporting and to arguable species concepts. Interpretation of available data is constrained by the morphological species concepts. The analysis suggests that there are not many species of heterotrophic flagellates (perhaps no more than 3000) and most have a cosmopolitan distribution. It is believed that there are assemblages of flagellates with distinctive taxonomic compositions, but it does not enable us to describe these more precisely until the impact factors external to the biology of the organisms but which influence our understanding of the structure of communities of heterotrophic flagellates is reduced. The model that emerges at the end of this work is that most productive aerated marine sediments are likely to contain 200 or more species of heterotrophic flagellates; that these species are likely to be found at similar locations world wide, and that their local abundance will be determined primarily by the grain size, by the productivity of the site, and whether than productivity is primary (algal) or secondary (bacterial).

Study on the Mechanism of Active Site Stabilization in Cold-Adapted PML

비나이쿠마 Daegu University, School of Biological Sciences 2017 국내박사

콜드 – 적응효소보다 따뜻한 온도의 활성효소와 비교하여 증가된 유연성을 나타낸다. 본 연구에서는 극성유기용매(DMSO및메탄올)가 효소의 유연성 및 안정성 관계에 미치는 영향을 조사하기 위해 유기용 제내성 저온 적응리파제PML을사용했습니다. 아크릴아마이드 유도된 담금질 및 열변성용융곡선을 사용하여 유기용매 존재하에서 PML의 모든 안정성에 대한 유연성을 결정하였다. 유기용매 농도가 증가함에 따라 효소는 증가된 유연성을 나타내지만 전반적인 안정성을 감소시켰다.그러나DMSO와 에탄올의 경우 최대 효소활성에 필요한 구조유연성이 다릅니다. 중온성 유기용 제내성 리파아제에서 중대한 구조 변화가 보고된 바 없으므로 본 연구는 저온 적응형 및 유기용 제내성 효소가 유기용매내에서 효소의 유연성과 안정성 관계를 연구하는 새로운 도구가 될 수있음을 시사한다. 효소의 안정성을 유지하는 요인을 조사하기 위해 우리는PML에서 보존된 이온상호작용을 확인했습니다.서열배열로부터 이는 중온성rPPE및 고온성 EstE1 효소에서도 또한 보존됨을 발견하였고, 따라서 이들 효소의 안정성을 유지하는데 있어서 이 이온상호작용의 역할을 연구하였다. 이온상호작용이 깨지면 모든 효소가 효소의 활성에 영향을 미치지 않으면 서열적 안정성을 잃어버렸다. 흥미롭게도 효소는 이온상호작용 파괴의 결과로 유기용매안정성을 잃어버렸다. 이결과는 이 위치에서 이온상호작용이 효소에 여분의 안정성을 제공함을 시사한다. 그러므로 3개의 멀리 떨어진 효소 모두에서 보존될 수 있다. Cold-adapted enzymes exhibit increased flexibility as compared with warmer temperature active enzymes. In this study, we used organic solvent-tolerant cold-adapted lipase PML, to investigate the effect of polar organic solvents (DMSO and methanol) on the flexibility and stability relationship of the enzyme. Using acrylamide-induced quenching and thermal denaturation melt curves we have determined the flexibility over all stability of PML in the presence of organic solvents. As the organic solvent concentration increases enzyme showed increased flexibility but it reduced its overall stability. However, the conformational flexibility required for maximal enzymatic activity is different for DMSO and ethanol. As no significant conformational changes have been reported from mesophilic organic solvent-tolerant lipases, our study suggests that cold-adapted and organic solvent-tolerant enzymes could be a novel tool to investigate the flexibility and stability relationship of the enzymes in organic solvent. To investigate the factors responsible for maintaining stability of the enzyme, we have identified conserved ionic interaction in PML. From the sequence alignment it was found this is also conserved in mesophilic rPPE and thermophilic EstE1enzymes, therefore we have investigated the role of this ionic interaction in maintaining stability of these enzymes. When the ionic interaction was broken all the enzymes lost their thermal stability without affecting activity of the enzymes. Interestingly enzymes also lost their organic solvent stability as the result of ionic interaction breaking. These results suggest that ionic interaction at this position provides extra stability to the enzyme. Therefore it may be conserved in all three distantly adopted enzymes.

Identification and characterization of a splice variant of the lysyl oxidase-like gene 3 (LOXL3)

이재은 Department of Biological Science Graduate School S 2005 국내박사

The lysyl oxidase-like gene 3 (LOXL3) is a member of the LOX family, encoding a copper-dependent amine oxidase that catalyzes the lysine-derived cross-links in the structural extracellular matrix (ECM) proteins, such as collagen and elastin. LOXL3 contains four scavenger receptor cysteine rich (SRCR) domains in the N-terminus in addition to the characteristic C-terminal domains of the LOX family, such as the copper-binding domain, the cytokine receptor-like (CRL) domain and the residues for the lysyl-tyrosyl quinone (LTQ) cofactor. Using a BLASTN search with the LOXL3 cDNA sequence, EST clones (BE302955 and BQ 424258) containing the cDNA sequence of LOXL3 from exon 4 to exon 14 but lacking the sequence corresponding to exon 5 of LOXL3 were identified. This LOXL3 splice variant (LOXL3-sv1) showed a distinct exon-intron structure from LOXL3, containing a 80 bp sequence corresponding to intron 3 of LOXL3 in the 5’UTR and a 561 bp sequence corresponding to the 3’ proximal genomic region of exon 14 in the 3’UTR. This novel variant of LOXL3 was expected to encode a polypeptide of 392 amino acids with a calculated molecular mass of 44 kDa containing only one SRCR domain and the characteristic domains of the LOX family. The LOXL3-sv1 protein showed amine oxidase activity toward elastin and various collagen types with substrate-specificity. In RT-PCR analysis with various human tissues, LOXL3-sv1 exhibited distinct patterns of expression from LOXL3 in different human tissues. Luciferase promoter assays revealed that the 5’ proximal region of exon 4 of LOXL3 showed a strong promoter activity, indicating that the LOXL3-sv1 mRNA is possibly expressed by a promoter element located in the 5’ proximal region of exon 4. To identify localization of LOXL3-sv1 in the extracellular matrix, immunostaining was performed in HEK 293 cells transfected with flag-tagged LOXL3-sv1. LOXL3-sv1 was visualized in cytoplasm and secreted into extracellular space. There was no detectable signal in nucleus. These results showed that LOXL3-sv1 is a secreted protein that functions as a substrate-specific amine oxidase. Taken together, these findings indicate that differential promoter activation leads to alternative expression of LOXL3 and LOXL3-sv1, both of which function as amine oxidases with different tissue specificity. Lysyl oxidase-like gene 3 (LOXL3)는 엘라스틴과 콜라젠과 같은 세포 외부 기질단백질의 교차결합을 촉진시키는 아민산화효소로써의 역할을 하는 LOX family 중의 하나이다. 이 단백질의 구조를 살펴보면, C-terminus에는 LOX family에 공통적으로 존재하는 copper-binding domain, cytokine receptor-like (CRL) domain, lysyl-tyrosyl quinone(LTQ) residues가 있으며 N-terminus부분에는 4개의 scavenger receptor cysteine rich (SRCR) domain들이 존재한다. 이런 LOXL3의 cDNA 염기서열을 가지고 EST database상에서 BLAST search를 통하여 LOXL3의 splice variant로 생각되는 두 개의 EST clone들 (BE302955 and BQ 424258)을 찾았다. 이 LOXL3의 splice variant (LOXL3-sv1)는 LOXL3의 4번에서 14번 exon들로 구성되어지며 5번 exon이 제외되어져 있다. 그리고 LOXL3의 intron 3에 해당하는 80 bp와 exon 14번의 3’end 방향으로 561 bp 크기의 염기서열을 추가로 transcription하고 있어서 LOXL3와는 다른 exon-intron구조를 나타낸다. LOXL3-sv1은 약 44 kDa으로 예상되는 분자적 질량을 가진 392 amino acids의 단백질을 합성하는데, C-terminus부분에는 LOX family에 공통적으로 존재하는 특징적인 domain들을 가지고 있으며, N-terminus에는 LOXL3가 4개의 SRCR domain들을 가지고 있는 반면, LOXL3-sv1은 단 한 개의 SRCR domain을 가진 구조를 보인다. 이 단백질의 아민산화효소로써의 역할을 확인하기 위한 실험에서, 엘라스틴과 여러 가지의 콜라젠에 대하여 기질 특이적인 활성을 가진 효소로의 기능을 확인할 수 있었다. 여러 가지 사람의 조직에서 RT-PCR 실험 방법을 이용하여 LOXL3와 LOXL3-sv1의 발현을 비교해 본 결과, 서로 다른 발현 패턴을 가짐을 알 수 있었다. 또한, Luciferase promoter assay를 통해 LOXL3-sv1이 LOXL3와는 다른 promoter 부분인, LOXL3의 4번 exon의 5’말단 방향에 위치하는 promoter 부분에 의하여 발현이 조절된다는 것을 확인하였다. 이런 사실들을 통해, LOXL3와는 다른 promoter 부분에 의하여, 아민산화효소로써의 기능을 하는 LOXL3-sv1이 조직 특이적 발현을 나타낸다는 것을 알 수 있었다. Flag-tagged LOXL3-sv1을 transfection시킨 HEK 293 cells에서 immuno-staining을 통해서 단백질이 세포질에서 발현되어서 세포 밖으로 분비되는 것을 확인하였다. 이 논문에서 LOXL3-sv1이 기질 특이성을 가진 아민산화효소로써 세포질 밖으로 분비되고, LOXL3와는 다른 promoter에 의해 조직 특이적인 발현 패턴을 갖는다는 것을 보여주고 있다.

Cyst Formation Mechanism by c-myc Overexpression in kidney of the Mxi1-deficient mouse

유경현 Department of Biological Science Graduate School S 2005 국내석사

The Mxi1 is a tumor-suppressor gene and essential roles in cellular growth control and in the induction and maintenance of the differentiated state. In addition, the Mxi1 is an antagonist of c-myc. The c-myc is well known to be oncogene. In previous study, c-myc is related with cyst formation disease like Autosomal Dominant Polycystic Kidney Disease (ADPKD). Overexpression pattern of c-myc is associated with increased proliferation and apoptosis of tubular epithelial cells might be similar to a molecular feature of early and late stages of human ADPKD. Based on these evidence, we investigated the cystogenesis mechanism involved in c-myc using the Mxi1-deficient mouse model. Especially it was reported that microscopic cysts were related with ADPKD and glomerulocystic kidney disease in the Mxi1-deficient mouse aged 7 months or more. We confirmed cyst formation, similar with cysts of renal disease including ADPKD, in kidney and liver through H&E staining. c-myc gene expression was increased in kidney of the Mxi1-deficient mouse using semi-quantitative RT-PCR. We confirmed to increase of proliferation and apoptosis in the Mxi1-deficient mouse. In addition, we monitored several genes that are related with renal cystic disease through semi-quantitative RT-PCR and immunohistochemical staining. Also, we screened and identified the Mxi1 related genes that are involved in cyst formation using cDNA microarray. These results showed that inactivation of the Mxi1 leads to renal cystic disease including ADPKD. Mxi1은 암억제유전자로써 세포 성장과 분화 과정 유도와 유지를 조절하는 데 필수적인 역할을 하는 유전자로 알려져 있다. 또한, Mxi1은 종양형성 유전자로 잘 알려진 c-myc과 antagonist이다. 이전 연구에서 c-myc은 Autosomal dominant polycystic kidney disease (ADPKD)에서 낭포 형성과 관련이 있다고 보고되고 있다. c-myc의 과발현은 세뇨관 상피세포의 증식과 세포사멸의 증가와 연관되어 있다. 이는 ADPKD 환자의 초기와 말기 단계에서 보여 지는 분자적 특성과 유사하다. 이것을 기본으로 하여, 우리는 Mxi1이 deficient 된 동물모델을 사용하여 c-myc이 포함되는 낭포형성 기전을 밝히고자 하였다. 선행 연구에서 7개월 이상 된 Mxi1-deficient 동물모델에서 ADPKD와 사구체 신염과 관련이 있는 미시적인 낭포를 발견하였다. 우리는 Hematoxylin & Eosin staining을 통하여 1년 된 Mxi1-deficient 동물모델에서 ADPKD의 낭포와 유사한 낭포들을 신장에서 발견하였으며, 간에서도 여러 낭포들이 발견됨을 확인하였다. 또한, semi-quantitative RT-PCR을 통하여 Mxi1 gene이 결여되었을 경우 c-myc이 과발현 되는 것을 확인하였다. 우리는 Mxi1-deficient 동물모델에서 어떠한 기전으로 낭포를 형성하는지 알아보고자, renal cystic disease에 관련되어 있는 몇 가지 유전자들의 발현정도를 semi-quantitative RT-PCR과 immunohistochemical staining을 통해 확인하였다. 또한 Mxi1-deficient 동물모델과 대조군 사이에 유전자 발현정도의 차이를 알아보고자 cDNA microarray를 사용하였다. 이러한 결과들은 Mxi1의 결여는 ADPKD를 포함하는 renal cystic disease를 초래한다는 것을 알 수 있다.