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

"

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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]

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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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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]

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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]

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1.	Gene. 2006 Apr 12;371(1):112-20. Epub 2006 Feb 14. Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri. Cheng Q, Hallmann A, Edwards L, Miller SM. Department of Biological Sciences, 1000 Hilltop Circle, University of Maryland, Baltimore County, Baltimore, MD 21250, USA. Abstract The green alga Volvox carteri possesses several thousand cells, but just two cell types: large reproductive cells called gonidia, and small, biflagellate somatic cells. Gonidia are derived from large precursor cells that are created during embryogenesis by asymmetric cell divisions. The J domain protein GlsA (Gonidialess A) is required for these asymmetric divisions and is believed to function with an Hsp70 partner. As a first step toward identifying this partner, we cloned and characterized V. carteri hsp70A, which is orthologous to HSP70A of the related alga Chlamydomonas reinhardtii. Like HSP70A, V. carteri hsp70A contains multiple heat shock elements (HSEs) and is highly inducible by heat shock. Consistent with these properties, Volvox transformants that harbor a glsA antisense transgene that is driven by an hsp70A promoter fragment express Gls phenotypes that are temperature-dependent. hsp70A appears to be the only gene in the genome that encodes a cytoplasmic Hsp70, so we conclude that Hsp70A is clearly the best candidate to be the chaperone that participates with GlsA in asymmetric cell division.
	PMID: 16476527 [PubMed - indexed for MEDLINE]

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1.	Protist. 2006 Oct;157(4):445-61. Epub 2006 Jul 18. Morphogenesis in the family Volvocaceae: different tactics for turning an embryo right-side out. Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany. armin.hallmann@gmx.de Abstract Green algae of the family Volvocaceae provide an unrivalled opportunity to analyze an evolutionary pathway leading from unicellularity to multicellularity with division of labor. One key step required for achieving multicellularity in this group was the development of a process for turning an embryo inside out: a morphogenetic process that is now known as "inversion," and that is a diagnostic feature of the group. Inversion is essential because at the end of its embryonic cleavage divisions, each volvocacean embryo contains all of the cells that will be present in an adult, but the flagellar ends of all cells are pointed toward the interior, rather than toward the exterior where they will need to be to function in locomotion. Inversion has been studied in greatest detail in Volvox carteri, but although all other volvocacean species have to struggle with the same awkward situation of being wrong-side out at the end of cleavage, they do it in rather different ways. Here, the inversion processes of six different volvocacean species (Gonium pectorale, Pandorina morum, Eudorina unicocca, Volvox carteri, Volvox tertius, and Volvox globator) are compared, in order to illustrate the variation in inversion patterns that exists within this family. The simplest inversion process occurs in the plate-shaped alga Gonium pectorale and the most complicated in the spherical alga Volvox globator. Gonium pectorale goes only from a concave-bowl shape to a slightly convex plate. In Volvox globator, the posterior hemisphere inverts completely before the anterior pole opens and the anterior hemisphere slides over the already-inverted posterior hemisphere; during both halves of this inversion process, the regions of maximum cell-sheet curvature move progressively, as radially symmetrical waves, along the posterior-anterior axis.
	PMID: 16854623 [PubMed - indexed for MEDLINE]

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1.	Science. 2007 Oct 12;318(5848):245-50. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Maréchal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, Dutcher S, Fernández E, Fukuzawa H, González-Ballester D, González-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV, Lohr M, Manuell A, Meier I, Mets L, Mittag M, Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV, Paulsen IT, Pazour G, Purton S, Ral JP, Riaño-Pachón DM, Riekhof W, Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN, Green P, Jorgensen R, Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo Y, Martínez D, Ngau WC, Otillar B, Poliakov A, Porter A, Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR. Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, CA 90095, USA. Abstract Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella. PMCID: PMC2875087 Free PMC Article
	PMID: 17932292 [PubMed - indexed for MEDLINE]

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1.	Development. 2006 Oct;133(20):4045-51. Epub 2006 Sep 13. Translational control of regA, a key gene controlling cell differentiation in Volvox carteri. Babinger K, Hallmann A, Schmitt R. Naturwissenschaftliche Fakultät III-Biologie und Vorklinische Medizin, University of Regensburg, D-93040 Regensburg, Germany. Abstract The complete division of labour between the reproductive and somatic cells of the green alga Volvox carteri is controlled by three types of genes. One of these is the regA gene, which controls terminal differentiation of the somatic cells. Here, we examined translational control elements located in the 5' UTR of regA, particularly the eight upstream start codons (AUGs) that have to be bypassed by the translation machinery before regA can be translated. The results of our systematic mutational, structural and functional analysis of the 5' UTR led us to conclude that a ribosome-shunting mechanism--rather than leaky scanning, ribosomal reinitiation, or internal ribosome entry site (IRES)-mediated initiation--controls the translation of regA mRNA. This mechanism, which involves dissociation of the 40S initiation complex from the message, followed by reattachment downstream, in order to bypass a secondary structure block in the mRNA, was validated by deleting the predicted ;landing site' (which prevented regA expression) and inserting a stable 64 nucleotide hairpin just upstream of this site (which did not prevent regA expression). We believe that this is the first report suggesting that translation of an mRNA in a green eukaryote is controlled by ribosome shunting. Free Article
	PMID: 16971469 [PubMed - indexed for MEDLINE]

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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]

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1.	Plant Signal Behav. 2008 Feb;3(2):124-7. VCRPs, small cysteine-rich proteins, might be involved in extracellular signaling in the green alga Volvox. Hallmann A. Department of Cellular and Developmental Biology of Plants; University of Bielefeld; Bielefeld, Germany. Abstract The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10(-16) M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism's environment to a reproductive cell must involve the ECM. Recently, a small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculated to be an extracellular second messenger from somatic cells to reproductive cells. Here a related protein, VCRP2, is presented, exhibiting a 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed. PMCID: PMC2634000 Free PMC Article
	PMID: 19704730 [PubMed - in process]

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1.	BMC Genomics. 2006 Dec 21;7:321. Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri. Nematollahi G, Kianianmomeni A, Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstr, 25, D-33615 Bielefeld, Germany. ghazaleh.nematollahi@uni-bielefeld.de <ghazaleh.nematollahi@uni-bielefeld.de> Abstract BACKGROUND: The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels. RESULTS: Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes. CONCLUSION: The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes. PMCID: PMC1774577 Free PMC Article
	PMID: 17184518 [PubMed - indexed for MEDLINE]

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1.	Plant Cell. 2008 Sep;20(9):2399-419. Epub 2008 Sep 12. A gender-specific retinoblastoma-related protein in Volvox carteri implies a role for the retinoblastoma protein family in sexual development. Kianianmomeni A, Nematollahi G, Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, D-33615 Bielefeld, Germany. Abstract Here, we describe the cloning and characterization of RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) from the green alga Volvox carteri. RBR1 expression increases substantially during embryogenesis and in response to the sex-inducer glycoprotein, but it decreases significantly under heat stress. While RBR1 is expressed in gonidia (asexual reproductive cells) and embryos, the largest proportion of RBR1 mRNA is found in parental somatic cells. The presence of 4 splice variants and 15 potential cyclin-dependent kinase phosphorylation sites suggests that RBR1 is subject to control at the posttranscriptional and posttranslational levels. Surprisingly, RBR1 is a gender-specific gene, mapping exclusively to the female mating-type locus. A procedure for stable nuclear transformation of males was established to generate RBR1-expressing males. These transformants exhibit enlarged reproductive cells, altered growth characteristics, and a prolonged embryogenesis. The results suggest that a functionally related analog of RBR1 exists in males. The reason for the divergent evolution of RBRs in females and males appears to be based on sexual development: males and females respond to the same sex-inducer with different cleavage programs and substantial differences in cellular differentiation. Thus, the gender-specific presence of RBR1 provides evidence for an additional, novel role for retinoblastoma family proteins in sexual development. PMCID: PMC2570726 Free PMC Article
	PMID: 18790828 [PubMed - indexed for MEDLINE]

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1.	Commun Integr Biol. 2009 Sep;2(5):396-9. Key elements of the retinoblastoma tumor suppressor pathway in Volvox carteri. Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, D-33615 Bielefeld, Germany. armin.hallmann@gmx.de Abstract The green alga Volvox carteri is one of the simplest multicellular organisms. It consists of only two cell types, somatic and reproductive cells, making it a suitable model system for studying cell division, multicellularity and cellular differentiation. Each of the approximately 2,000-4,000 cells of an adult, asexual organism arises through a sequence of symmetric and asymmetric cleavage divisions from a single, asexual reproductive cell. As in ontogenetic development of higher organisms, the fate of a Volvox blastomere (i.e., whether it undergoes division or differentiation) is determined by a complex balance of regulators. Retinoblastomarelated proteins (RBRs) seem to act as key regulators and hubs in cell cycle control and, therefore, have been investigated in detail in higher organisms. Recently, the identification and characterization of a gender-specific RBR in Volvox, RBR1, revealed a role for the retinoblastoma protein family in sexual development. RBRs are elements of a conserved signal-transduction pathway called the retinoblastoma (RB) tumor suppressor pathway. In addition to RBR1, other key components of this pathway are present in Volvox, demonstrating that the RB signal-transduction pathway is utilized by these simple green algae. PMCID: PMC2775231 Free PMC Article
	PMID: 19907698 [PubMed]

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1.	BMC Biotechnol. 2009 Jul 10;9:64. Stable nuclear transformation of Gonium pectorale. Lerche K, Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstr, 25, D-33615 Bielefeld, Germany. kai.lerche@gmx.de Abstract BACKGROUND: Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of approximately 16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri. RESULTS: Stable nuclear transformation of G. pectorale was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (aphVIII) of Streptomyces rimosus, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from Chlamydomonas reinhardtii or from Volvox carteri. After particle gun bombardment of wild type Gonium cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of approximately 30-50% when cells were co-bombarded with aphVIII-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod Gaussia princeps, which was previously engineered to match the codon usage in C. reinhardtii, Gonium transformants show luciferase activity through light emission in bioluminescence assays. CONCLUSION: Flanking sequences that include promoters from C. reinhardtii and from V. carteri work in G. pectorale and allow the functional expression of heterologous genes, such as the selectable marker gene aphVIII of S. rimosus or the co-transformed, codon-optimized G. princeps luciferase gene, which turned out to be a suitable reporter gene in Gonium. The availability of a method for transformation of Gonium makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular Chlamydomonas, the 16-celled Gonium, and the multicellular Volvox. PMCID: PMC2720962 Free PMC Article
	PMID: 19591675 [PubMed - indexed for MEDLINE]

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1.	J Biotechnol. 2007 Aug 1;131(1):27-33. Epub 2007 May 24. Functional integration of the HUP1 hexose symporter gene into the genome of C. reinhardtii: Impacts on biological H(2) production. Doebbe A, Rupprecht J, Beckmann J, Mussgnug JH, Hallmann A, Hankamer B, Kruse O. Department of Biology, Algae Biotech Group, University Bielefeld, Bielefeld, Germany. Abstract Phototrophic organisms use photosynthesis to convert solar energy into chemical energy. In nature, the chemical energy is stored in a diverse range of biopolymers. These sunlight-derived, energy-rich biopolymers can be converted into environmentally clean and CO(2) neutral fuels. A select group of photosynthetic microorganisms have developed the ability to extract and divert protons and electrons derived from water to chloroplast hydrogenase(s) to produce molecular H(2) fuel. Here, we describe the development and characterization of C. reinhardtii strains, derived from the high H(2) production mutant Stm6, into which the HUP1 (hexose uptake protein) hexose symporter from Chlorella kessleri was introduced. The isolated cell lines can use externally supplied glucose for heterotrophic growth in the dark. More importantly, external glucose supply (1mM) was shown to increase the H(2) production capacity in strain Stm6Glc4 to approximately 150% of that of the high-H(2) producing strain, Stm6. This establishes the foundations for a new fuel production process in which H(2)O and glucose can simultaneously be used for H(2) production. It also opens new perspectives on future strategies for improving bio-H(2) production efficiency under natural day/night regimes and for using sugar waste material for energy production in green algae as photosynthetic catalysts.
	PMID: 17624461 [PubMed - indexed for MEDLINE]

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1.	Plant Physiol. 2009 Sep;151(1):347-66. Epub 2009 Jul 29. Channelrhodopsins of Volvox carteri are photochromic proteins that are specifically expressed in somatic cells under control of light, temperature, and the sex inducer. Kianianmomeni A, Stehfest K, Nematollahi G, Hegemann P, Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, 33615 Bielefeld, Germany. Abstract Channelrhodopsins are light-gated ion channels involved in the photoresponses of microalgae. Here, we describe the characterization of two channelrhodopsins, Volvox channelrhodopsin-1 (VChR1) and VChR2, from the multicellular green alga Volvox carteri. Both are encoded by nuclear single copy genes and are highly expressed in the small biflagellated somatic cells but not in the asexual reproductive cells (gonidia). Expression of both VChRs increases after cell cleavage and peaks after completion of embryogenesis, when the biosynthesis of the extracellular matrix begins. Likewise, expression of both transcripts increases after addition of the sex-inducer protein, but VChR2 is induced much more than VChR1. The expression of VChR1 is specifically promoted by extended dark periods, and heat stress reduces predominantly VChR1 expression. Expression of both VChRs increased under low light conditions, whereas cold stress and wounding reduced expression. Both VChRs were spectroscopically studied in their purified recombinant forms. VChR2 is similar to the ChR2 counterpart from Chlamydomonas reinhardtii with respect to its absorption maximum (460 nm) and photocycle dynamics. In contrast, VChR1 absorbs maximally at 540 nm at low pH (D540), shifting to 500 nm at high pH (D500). Flash photolysis experiments showed that after light excitation, the D540 dark state bleaches and at least two photoproducts, P600 and P500, are sequentially populated during the photocycle. We hypothesize that VChR2 is a general photoreceptor that is responsible for the avoidance of blue light and might play a key role in sexual development, whereas VChR1 is the main phototaxis photoreceptor under vegetative conditions, as it is more specifically adapted to environmental conditions and the developmental stages of Volvox. PMCID: PMC2736010 [Available on 2010/9/1]
	PMID: 19641026 [PubMed - indexed for MEDLINE]

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1.	Plant J. 2006 Jan;45(2):292-307. The pherophorins: common, versatile building blocks in the evolution of extracellular matrix architecture in Volvocales. Hallmann A. Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstr. 25, D-33615 Bielefeld, Germany. armin.hallmann@gmx.de Abstract Green algae of the order Volvocales provide an unrivalled opportunity for exploring the transition from unicellularity to multicellularity. They range from unicells, like Chlamydomonas, through homocytic colonial forms with increasing cooperation of individual cells, like Gonium or Pandorina, to heterocytic multicellular forms with different cell types and a complete division of labour, like Volvox. A fundamental requirement for the evolution of multicellularity is the development of a complex, multifunctional extracellular matrix (ECM). The ECM has many functions, which can change under developmental control or as a result of environmental factors. Here molecular data from 15 novel proteins are presented. These proteins have been identified in Chlamydomonas reinhardtii, Gonium pectorale, Pandorina morum and Volvox carteri, and all belong to a single protein family, the pherophorins. Pherophorin-V1 is shown to be a glycoprotein localized to the 'cellular zone' of the V. carteri ECM. Pherophorin-V1 and -V2 mRNAs are strongly induced not only by the sex inducer, which triggers sexual development at extremely low concentrations, but also by mechanical wounding. Like the extensins of higher plants, which are also developmentally controlled or sometimes inducible by wounding, the pherophorins contain a (hydroxy-)proline-rich (HR) rod-like domain and are abundant within the extracellular compartment. In contrast to most extensins, pherophorins have additional globular A and B domains on both ends of the HR domains. Therefore pherophorins most closely resemble a particular class of higher plant extensin, the solanaceous lectins (e.g. potato lectin), suggesting multivalent carbohydrate-binding functions are present within the A and B domains and are responsible for cross-linking. Our results suggest that pherophorins are used as the building blocks for the extracellular scaffold throughout the Volvocales, with the characteristic mesh sizes in different ECM structures being a result of the highly diverse extensions of the HR domains. Pherophorins have therefore been a versatile element during the evolution of ECM architecture in these green algae.
	PMID: 16367971 [PubMed - indexed for MEDLINE]

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1.	BMC Biol. 2010 Jul 27;8(1):103. [Epub ahead of print] How 5000 independent rowers coordinate their strokes in order to row into the sunlight: Phototaxis in the multicellular green alga Volvox. Ueki N, Matsunaga S, Inouye I, Hallmann A. Abstract ABSTRACT: BACKGROUND: The evolution of multicellular motile organisms from unicellular ancestors required the utilization of previously evolved tactic behavior in a multicellular context. Volvocine green algae are uniquely suited for studying tactic responses during the transition to multicellularity because they range in complexity from unicellular to multicellular genera. Phototactic responses are essential for these flagellates because they need to orientate themselves to receive sufficient light for photosynthesis, but how does a multicellular organism accomplish phototaxis without any known direct communication among cells? Several aspects of the photoresponse have previously been analyzed in volvocine algae, particularly in the unicellular alga Chlamydomonas. RESULTS: In this study, the phototactic behavior in the spheroidal, multicellular volvocine green alga Volvox rousseletii (Volvocales, Chlorophyta) was analyzed. In response to light stimuli, not only did the flagella waveform and beat frequency change, but the effective stroke was reversed. Moreover, there was a photoresponse gradient from the anterior to the posterior pole of the spheroid, and only cells of the anterior hemisphere showed an effective response. The latter caused a reverse of the fluid flow that was confined to the anterior hemisphere. The responsiveness to light is consistent with an anterior-to-posterior size gradient of eyespots. At the posterior pole, the eyespots are tiny or absent, making the corresponding cells appear to be blind. Pulsed light stimulation of an immobilized spheroid was used to simulate the light fluctuation experienced by a rotating spheroid during phototaxis. The results demonstrated that in free-swimming spheroids, only those cells of the anterior hemisphere that face toward the light source reverse the beating direction in the presence of illumination; this behavior results in phototactic turning. Moreover, positive phototaxis is facilitated by gravitational forces. Under our conditions, V. rousseletii spheroids showed no negative phototaxis. CONCLUSIONS: On the basis of our results, we developed a mechanistic model that predicts the phototactic behavior in V. rousseletii. The model involves photoresponses, periodically changing light conditions, morphological polarity, rotation of the spheroid, two modes of flagellar beating, and the impact of gravity. Our results also indicate how recently evolved multicellular organisms adapted the phototactic capabilities of their unicellular ancestors to multicellular life. Free Article
	PMID: 20663212 [PubMed - as supplied by publisher]