Effective molecular examination of eukaryotic plankton species diversity in environmental seawater using environmental PCR, PCR-RFLP, and sequencing

Effective molecular examination of eukaryotic plankton species diversity in environmental seawater using environmental PCR, PCR-RFLP, and sequencing: "

Abstract

Phytoplankton are primary producers and can be important indicators of environmental change. To monitor the plankton species
composition of environmental seawater samples, we developed a molecular method composed of colony polymerase chain reaction
(PCR), polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and sequencing. A clone library of the
ribosomal small subunit RNA gene (18S rDNA) in the nuclear genome was constructed by environmental PCR using a newly designed
primer set and clones were directly amplified by colony PCR. To select unique putative clones, we choose a PCR-RFLP method
that employed two restriction enzymes (MseI and Tsp509I). After the PCR-RFLP pattern was evaluated, selected clones were sequenced and analyzed. In this study, we revealed the
hidden biodiversity in environmental seawater containing a wide range of taxonomic groups in the Alveolata (Ciliphora and
Dinophyceae), Euglenozoa, Stramenopiles (Bacillariophyta), and Viridiplantae (Chlorophyta) without the need to conduct extensive
colony isolation techniques. Moreover, we found species of fungi and Metazoa (Arthropoda, Annelida, and Mollusca). Therefore,
this improved molecular method can be used to generate a robust database describing the species diversity of environmental
samples and provide useful information regarding the dynamics of the eukaryotic plankton community structure.

• Content Type Journal Article
• DOI 10.1007/s10811-010-9509-7
• Authors
  
  
  • Sang-Rae Lee, Pusan National University Marine Research Institute Busan 609-735 South Korea
  • Jung Hyun Oak, Pusan National University Marine Research Institute Busan 609-735 South Korea
  • Ik Kyo Chung, Pusan National University Division of Earth Environmental System Busan 609-735 South Korea
  • Jin Ae Lee, Inje University School of Environmental Science and Engineering Gimhae 621-749 South Korea
  
  

• Journal Journal of Applied Phycology
• Online ISSN 1573-5176
• Print ISSN 0921-8971

"

High-quality RNA preparation for cDNA library construction of the Antarctic sea–ice alga Chlamydomonas sp. ICE-L

High-quality RNA preparation for cDNA library construction of the Antarctic sea–ice alga Chlamydomonas sp. ICE-L: "

Abstract

To study the molecular mechanism of the Antarctic sea–ice alga in adaptation to polar sea–ice environments, the RNA was prepared
for cDNA library construction of Chlamydomonas sp. ICE-L. Three different methods were tested to prepare total RNA from this psychrophilic, unicellular green alga rich
in protein and polysaccharide. Lauryl sodium sulfate- based method allowed a most effective extraction of high-quality total
RNA compared to the other methods. Total RNA extracted with this protocol was used for cDNA library construction. The recombination
rate of constructed cDNA library was 98.60%, the primary titer was 7.15 × 10⁶ pfu, and an average sequence length was 1.2 kb. These results show that with a high-quality RNA preparation, a cDNA library
can be constructed successfully for Chlamydomonas sp. ICE-L.

• Content Type Journal Article
• DOI 10.1007/s10811-010-9519-5
• Authors
  
  
  • Guangting Wu, State Oceanic Administration Key Laboratory of Marine Bioactive Substances 266061 Qingdao China
  • Chenlin Liu, State Oceanic Administration Key Laboratory of Marine Bioactive Substances 266061 Qingdao China
  • Shenghao Liu, State Oceanic Administration Key Laboratory of Marine Bioactive Substances 266061 Qingdao China
  • Bailin Cong, State Oceanic Administration Key Laboratory of Marine Bioactive Substances 266061 Qingdao China
  • Xiaohang Huang, State Oceanic Administration Key Laboratory of Marine Bioactive Substances 266061 Qingdao China
  
  

• Journal Journal of Applied Phycology
• Online ISSN 1573-5176
• Print ISSN 0921-8971

"

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Ulva pertusa (Ulvophyceae, Chlorophyta)

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Ulva pertusa (Ulvophyceae, Chlorophyta): "

Abstract

In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone a full-length cytosolic
heat shock protein 70 of Ulva pertusa (designated as UPHsp70). Bioinformatics was used to analyze structural features, homologous relationship, and phylogenetic
position of UPHsp70. The full length of UPHsp70 cDNA was 2,283 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated
region of 247 bp, and an open reading frame of 1,971 bp encoding a polypeptide of 656 amino acids with an estimated molecular
weight of 71.13 kDa and an estimated isoelectric point of 5.04. The UPHsp70 had four degenerate repeats of tetrapeptide GGMP
and three typical Hsp70 signature motifs. The specific C-terminus amino acid sequence of cytosolic UPHsp70 was EEVD, and the
conservation of Hsp70s of N-terminus was higher than that of C-terminus. The homology between UPHsp70 and Hsp70s of other
known algae and land plants was more than 70%. Under different stress conditions, mRNA expression levels of UPHsp70 were quantified
by quantitative reverse transcriptase-polymerase chain reaction. When U. pertusa samples were kept in different temperatures (5–40°C) for 1 h, the expression level of UPHsp70 at 5°C, 35°C, or 40°C was over
onefold higher than that at 25°C. When U. pertusa samples were kept at 30°C for different times (0–12 h), the mRNA expression level of UPHsp70 had a trend of rise first then
fall. The expression level of UPHsp70 reached maximum level after 5 h. When U. pertusa samples were kept in different salt concentrations (0–45‰) for 2 h, the expression level of UPHsp70 at 0‰ or 5‰ salt concentration
was twofold higher than that at 30‰ for 2 h. The expression levels of UPHsp70 at 30‰, 35‰, and 40‰ were low and had no difference
(P < 0.05). When U. pertusa samples were kept at ultraviolet irradiation or desiccated for different times (0–4 h), the mRNA expression level of UPHsp70
reached maximum level after 3.0 h; after that, it was maintained at high level.

• Content Type Journal Article
• DOI 10.1007/s10811-010-9561-3
• Authors
  
  
  • Wandong Fu, Zhejiang Marine Development Research Institute Zhoushan 316100 People’s Republic of China
  • Li Shuai, Qingdao University College of Chemistry, Chemical Engineering and Environmental Science Qingdao 266071 People’s Republic of China
  • Jianting Yao, Chinese Academy of Sciences Institute of Oceanology Qingdao 266071 People’s Republic of China
  • Bin Zheng, Zhejiang Marine Development Research Institute Zhoushan 316100 People’s Republic of China
  • Mingjie Zhong, Zhejiang Marine Development Research Institute Zhoushan 316100 People’s Republic of China
  • Delin Duan, Chinese Academy of Sciences Institute of Oceanology Qingdao 266071 People’s Republic of China
  
  

• Journal Journal of Applied Phycology
• Online ISSN 1573-5176
• Print ISSN 0921-8971

"

PubMed Search Results

PubMed Results

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1.	J Biol Chem. 2002 Aug 2;277(31):28280-6. Epub 2002 May 28. An extracellular matrix-localized metalloproteinase with an exceptional QEXXH metal binding site prefers copper for catalytic activity. Heitzer M, Hallmann A. Lehrstuhl Biochemie I, Universität Regensburg, D-93053 Regensburg, Germany. Abstract The extracellular matrix (ECM) of the simple multicellular organism Volvox contains many region-specific morphological elements and mediates a variety of developmental and physiological responses by modification of its components. The fact that >95% of the mature organism is ECM makes Volvox suitable as a model system for ECM investigations. VMPs are a family of Volvox genes that are homologous to zinc-dependent matrix metalloproteinases (MMPs). Here we describe the identification and purification of the first VMP protein, VMP3. The 470-kDa VMP3 glycoprotein is localized within the ECM, and its biosynthesis is induced by the sex pheromone. The metal binding motif of VMP3 is QEXXH, not HEXXH as known for approximately 1300 other metalloproteinases. VMP3 shows proteinase activity and is inhibited by EDTA or the MMP inhibitor GM 6001, but in contrast to all known proteinases, VMP3 clearly prefers copper for activity rather than zinc. The exchange from Q to H within the QEXXH motif abolishes its copper preference. The unique properties of VMP3 suggest a novel type of metalloproteinase. Free Article
	PMID: 12034745 [PubMed - indexed for MEDLINE]

PubMed Search Results

PubMed Results

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1.	Int Rev Cytol. 2003;227:131-82. Extracellular matrix and sex-inducing pheromone in Volvox. Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, D-33615 Bielefeld, Germany. Abstract During evolution of multicellularity it was imperative to create a complex, multifunctional extracellular matrix (ECM) out of the simple cell wall of a unicellular ancestor. The green alga Volvox represents one of the simplest multicellular organisms, but even so, it already has a highly developed ECM. This ECM is mainly composed of an assortment of glycoproteins, many of which are hydroxyproline rich and extensively sulfated. Several ECM proteins are cross-linked and might have only structural functions. However, the ECM does not represent a static but rather a dynamic and multifunctional interface between a cell and its neighboring cells or its environment. It not only provides protection and structural support for the shape of each cell and the organism as a whole, but also plays a broad range of biological roles in growth, development, reproduction, and responses to environmental stress or wounding. The variety of functions of the ECM requires many glycoproteins to do the work. To attain a high flexibility and adaptability, almost all ECM glycoproteins from Volvox consist of modules, defined as functional subunits that form modular mosaic proteins with an outstanding combinatorial potential. The ECM's functions are not only extensive but also change under developmental control or by environmental incidents. The changing scope of duties necessitates a permanent ECM turnover and remodeling. In Volvox carteri one particularly challenging trigger of such ECM modifications is a sex-inducing pheromone, which is one of the most potent biological effector molecules known: the glycoprotein pheromone is fully effective for inducing sexual development in males and females at concentrations as low as 10(-16) M. The earliest detectable response to the pheromone is the synthesis of ECM glycoproteins.
	PMID: 14518551 [PubMed - indexed for MEDLINE]

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PubMed Results

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1.	Science. 2010 Jul 9;329(5988):223-6. Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Prochnik SE, Umen J, Nedelcu AM, Hallmann A, Miller SM, Nishii I, Ferris P, Kuo A, Mitros T, Fritz-Laylin LK, Hellsten U, Chapman J, Simakov O, Rensing SA, Terry A, Pangilinan J, Kapitonov V, Jurka J, Salamov A, Shapiro H, Schmutz J, Grimwood J, Lindquist E, Lucas S, Grigoriev IV, Schmitt R, Kirk D, Rokhsar DS. U.S. Department of Energy, Joint Genome Institute, Walnut Creek, CA 94598, USA. Comment in: Science. 2010 Jul 9;329(5988):128-9. Abstract The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-mega-base pair genome of V. carteri and compared its approximately 14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal-specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.
	PMID: 20616280 [PubMed - indexed for MEDLINE]

PubMed Search Results

PubMed Results

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1.	Planta. 2007 Aug;226(3):719-27. Epub 2007 Apr 13. A small cysteine-rich extracellular protein, VCRP, is inducible by the sex-inducer of Volvox carteri and by wounding. Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany. armin.hallmann@gmx.de Abstract The green alga Volvox carteri represents one of the simplest multicellular organisms: it is composed of only two cell types, somatic and reproductive. Volvox is capable of both vegetative and sexual reproduction. Sexual development of males and females is triggered by a sex-inducer at concentrations as low as 10(-16) M. By differential screenings of cDNA libraries, a novel gene was identified that is under the control of this sex-inducer and that encodes a small cysteine-rich extracellular protein, named VCRP. Analysis of the VCRP polypeptide sequence suggests ten disulfide bonds and a dimetal-binding capacity. VCRP mRNA is detectable in males and females approximately 1 h after the spheroids' first contact with the sex-inducer, but transcription is restricted to the somatic cell-type. mRNA and protein synthesis is triggered not only by the sex-inducer, but also by wounding. VCRP does not share significant sequence similarity with any known protein sequence, but a potential EGF-like calcium-binding motif and a potential plant metallothionein family-15 motif have been identified. The characteristics of VCRP suggest a function as a signal transducer molecule, an extracellular second messenger from somatic cells to reproductive cells, or a role within the stress response.
	PMID: 17431666 [PubMed - indexed for MEDLINE]