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Armin Hallmann - One of the best experts on this subject based on the ideXlab platform.
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Stable Nuclear Transformation of Pandorina morum
BMC Biotechnology, 2014Co-Authors: Kai Lerche, Armin HallmannAbstract:Background: Volvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8–16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation, morphogenesis and epithelial folding, sexual reproduction and evolution of multicellularity. Pandorina is the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction. So far, molecular-biological investigations in P. morum were constricted due to the absence of methods for Transformation of this species, which is a prerequisite for introduction of reporter genes and (modified) genes of interest. Results: Stable Nuclear Transformation of P. morum was achieved using chimeric constructs with a selectable marker, a reporter gene, promoters and upstream and downstream flanking sequences from heterologous sources. DNA was introduced into the cells by particle bombardment with plasmid-coated gold particles. The aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus under control of an artificial, heterologous promoter was used as the selectable marker. The artificial promoter contained a tandem arrangement of the promoter of both the heat shock protein 70A (hsp70A) and the ribulose-1,5-bisphosphat-carboxylase/-oxygenase S3 (rbcS3) gene of Volvox carteri. Due to the expression of aphVIII, transformants gained up to 333-fold higher resistance to paromomycin in comparison to the parent wild-type strain. The heterologous luciferase (gluc )g ene ofGaussia princeps, which was previously genetically engineered to match the Nuclear codon usage of Chlamydomonas reinhardtii, was used as a co-transformed, unselectable reporter gene. The expression of the co-bombarded gluc gene in transformants and the induction of gluc by heat shock were demonstrated through bioluminescence assays. Conclusion: Stable Nuclear Transformation of P. morum using the particle bombardment technique is now feasible. Functional expression of heterologous genes is achieved using heterologous flanking sequences from Volvox carteri and Chlamydomonas reinhardtii .T heaphVIII gene of the actinobacterium S. rimosus can be used as a selectable marker for Transformation experiments in the green alga P. morum .T hegluc gene of the marine copepod G. princeps, expressed under control of heterologous promoter elements, represents a suitable reporter gene for monitoring gene expression or for other applications in P. morum.
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Stable Nuclear Transformation of Eudorina elegans
BMC Biotechnology, 2013Co-Authors: Kai Lerche, Armin HallmannAbstract:Background A fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga ( Chlamydomonas reinhardtii ) and a multicellular alga with differentiated cell types ( Volvox carteri ). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans , which is composed of 16–32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina , and only limited DNA and/or protein sequence information is available. Results Here, we describe the stable Nuclear Transformation of E. elegans by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3^′-phosphotransferase VIII ( aph VIII) gene of Streptomyces rimosus , an actinobacterium, under the control of an artificial promoter consisting of two V. carteri promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-Transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase ( gluc ) gene from the marine copepod Gaussia princeps , which previously was engineered to match the codon usage of C. reinhardtii , was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri . Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded gluc gene was demonstrated by transcription analysis and bioluminescence assays. Conclusions Heterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable Nuclear Transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.
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Stable Nuclear Transformation of Eudorina elegans
BMC Biotechnology, 2013Co-Authors: Kai Lerche, Armin HallmannAbstract:A fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga (Chlamydomonas reinhardtii) and a multicellular alga with differentiated cell types (Volvox carteri). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans, which is composed of 16–32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina, and only limited DNA and/or protein sequence information is available. Here, we describe the stable Nuclear Transformation of E. elegans by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus, an actinobacterium, under the control of an artificial promoter consisting of two V. carteri promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-Transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase (gluc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of C. reinhardtii, was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri. Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded gluc gene was demonstrated by transcription analysis and bioluminescence assays. Heterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable Nuclear Transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.
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Stable Nuclear Transformation of Gonium pectorale
BMC Biotechnology, 2009Co-Authors: Kai Lerche, Armin HallmannAbstract: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 ~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.
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a gender specific retinoblastoma related protein in volvox carteri implies a role for the retinoblastoma protein family in sexual development
The Plant Cell, 2008Co-Authors: Arash Kianianmomeni, Ghazaleh Nematollahi, Armin HallmannAbstract: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.
Kai Lerche - One of the best experts on this subject based on the ideXlab platform.
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Stable Nuclear Transformation of Pandorina morum
BMC Biotechnology, 2014Co-Authors: Kai Lerche, Armin HallmannAbstract:Background: Volvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8–16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation, morphogenesis and epithelial folding, sexual reproduction and evolution of multicellularity. Pandorina is the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction. So far, molecular-biological investigations in P. morum were constricted due to the absence of methods for Transformation of this species, which is a prerequisite for introduction of reporter genes and (modified) genes of interest. Results: Stable Nuclear Transformation of P. morum was achieved using chimeric constructs with a selectable marker, a reporter gene, promoters and upstream and downstream flanking sequences from heterologous sources. DNA was introduced into the cells by particle bombardment with plasmid-coated gold particles. The aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus under control of an artificial, heterologous promoter was used as the selectable marker. The artificial promoter contained a tandem arrangement of the promoter of both the heat shock protein 70A (hsp70A) and the ribulose-1,5-bisphosphat-carboxylase/-oxygenase S3 (rbcS3) gene of Volvox carteri. Due to the expression of aphVIII, transformants gained up to 333-fold higher resistance to paromomycin in comparison to the parent wild-type strain. The heterologous luciferase (gluc )g ene ofGaussia princeps, which was previously genetically engineered to match the Nuclear codon usage of Chlamydomonas reinhardtii, was used as a co-transformed, unselectable reporter gene. The expression of the co-bombarded gluc gene in transformants and the induction of gluc by heat shock were demonstrated through bioluminescence assays. Conclusion: Stable Nuclear Transformation of P. morum using the particle bombardment technique is now feasible. Functional expression of heterologous genes is achieved using heterologous flanking sequences from Volvox carteri and Chlamydomonas reinhardtii .T heaphVIII gene of the actinobacterium S. rimosus can be used as a selectable marker for Transformation experiments in the green alga P. morum .T hegluc gene of the marine copepod G. princeps, expressed under control of heterologous promoter elements, represents a suitable reporter gene for monitoring gene expression or for other applications in P. morum.
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Stable Nuclear Transformation of Eudorina elegans
BMC Biotechnology, 2013Co-Authors: Kai Lerche, Armin HallmannAbstract:Background A fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga ( Chlamydomonas reinhardtii ) and a multicellular alga with differentiated cell types ( Volvox carteri ). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans , which is composed of 16–32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina , and only limited DNA and/or protein sequence information is available. Results Here, we describe the stable Nuclear Transformation of E. elegans by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3^′-phosphotransferase VIII ( aph VIII) gene of Streptomyces rimosus , an actinobacterium, under the control of an artificial promoter consisting of two V. carteri promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-Transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase ( gluc ) gene from the marine copepod Gaussia princeps , which previously was engineered to match the codon usage of C. reinhardtii , was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri . Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded gluc gene was demonstrated by transcription analysis and bioluminescence assays. Conclusions Heterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable Nuclear Transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.
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Stable Nuclear Transformation of Eudorina elegans
BMC Biotechnology, 2013Co-Authors: Kai Lerche, Armin HallmannAbstract:A fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga (Chlamydomonas reinhardtii) and a multicellular alga with differentiated cell types (Volvox carteri). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans, which is composed of 16–32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina, and only limited DNA and/or protein sequence information is available. Here, we describe the stable Nuclear Transformation of E. elegans by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus, an actinobacterium, under the control of an artificial promoter consisting of two V. carteri promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-Transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase (gluc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of C. reinhardtii, was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri. Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded gluc gene was demonstrated by transcription analysis and bioluminescence assays. Heterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable Nuclear Transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.
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Stable Nuclear Transformation of Gonium pectorale
BMC Biotechnology, 2009Co-Authors: Kai Lerche, Armin HallmannAbstract: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 ~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.
Henry Daniell - One of the best experts on this subject based on the ideXlab platform.
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the complete chloroplast genome sequence of citrus sinensis l osbeck var ridge pineapple organization and phylogenetic relationships to other angiosperms
BMC Plant Biology, 2006Co-Authors: Michael G Bausher, Nameirakpam D Singh, Robert K Jansen, Henry DaniellAbstract:Background The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and Nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over Nuclear Transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms.
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metabolic engineering of the chloroplast genome using the echerichia coli ubic gene reveals that chorismate is a readily abundant plant precursor for p hydroxybenzoic acid biosynthesis
Plant Physiology, 2004Co-Authors: Paul V Viitanen, Andrew L Devine, Muhammad S Khan, Deborah L Deuel, Drew E Van Dyk, Henry DaniellAbstract:p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5′ untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for Nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for Nuclear Transformation. These experiments demonstrate that the current limitation for pHBA production in Nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid Transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.
P De Boer - One of the best experts on this subject based on the ideXlab platform.
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Incomplete Nuclear Transformation of human spermatozoa in oligo-astheno-teratospermia: characterization by indirect immunofluorescence of chromatin and thiol status.
'Oxford University Press (OUP)', 2008Co-Authors: Ramos L., Heijden, G.w. Van Der, Derijck A.h.a., Berden J.h.m., Kremer J.a.m., Vlag J. Van Der, P De BoerAbstract:Contains fulltext : 69766.pdf (publisher's version ) (Open Access)BACKGROUND Sperm heterogeneity in the human, as observed in oligo-astheno-teratozoospermia (OAT), is associated with hypospermatogenesis. METHODS The chromatin of sperm from OAT and normospermic males was characterized with antibodies specific for nucleosomes, the histone H3.1/H3.2 isoform, histone TH2B, apoptosis-associated H4 acetylation (KM-2) and protamines. Subsequently, sperm samples were stained with the thiol-specific fluorochrome monobromobimane (mBBr) before and after reduction with dithiotreitol (DTT) as most thiol groups reside in the cysteine-rich protamines. We also used fluorescence-activated cell sorter (FACS) for sperm histograms and sorting for high or low free and total thiol levels. These fractions were further analysed for DNA damage with the TdT-UTP nick end-labelling (TUNEL) assay. RESULTS OAT sperm nuclei stained higher for nucleosomes and KM2-epitopes, and lower for TH2B. For free, and total, thiol groups, OAT sperm were characterized by biphasic distributions, reflecting incomplete thiol oxidation as well as overoxidation, and possibly reduced protamine contents. The TUNEL assay on sperm subfractions, for both control and OAT sperm, revealed that a lower level of free thiol groups is associated with a higher TUNEL incidence, and this relationship was also found for total thiol levels. Hence, both overoxidation and a low total number of thiol groups predestine for sperm apoptosis. CONCLUSIONS Chromatin characteristics reflecting an incomplete nucleosome to protamine remodelling were found in subfertile males. Sperm apoptosis is related to both incomplete remodelling and protamine overoxidation
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incomplete Nuclear Transformation of human spermatozoa in oligo astheno teratospermia characterization by indirect immunofluorescence of chromatin and thiol status
Human Reproduction, 2007Co-Authors: L Ramos, G W Van Der Heijden, Alwin A H A Derijck, Jo H M Berden, John Kremer, J Van Der Vlag, P De BoerAbstract:BACKGROUND: Sperm heterogeneity in the human, as observed in oligo-astheno-teratozoospermia (OAT), is associated with hypospermatogenesis. METHODS: The chromatin of sperm from OAT and normospermic males was characterized with antibodies specific for nucleosomes, the histone H3.1/H3.2 isoform, histone TH2B, apoptosisassociated H4 acetylation (KM-2) and protamines. Subsequently, sperm samples were stained with the thiol-specific fluorochrome monobromobimane (mBBr) before and after reduction with dithiotreitol (DTT) as most thiol groups reside in the cysteine-rich protamines. We also used fluorescence-activated cell sorter (FACS) for sperm histograms and sorting for high or low free and total thiol levels. These fractions were further analysed for DNA damage with the TdT-UTP nick end-labelling (TUNEL) assay. RESULTS: OAT sperm nuclei stained higher for nucleosomes and KM2-epitopes, and lower for TH2B. For free, and total, thiol groups, OAT sperm were characterized by biphasic distributions, reflecting incomplete thiol oxidation as well as overoxidation, and possibly reduced protamine contents. The TUNEL assay on sperm subfractions, for both control and OAT sperm, revealed that a lower level of free thiol groups is associated with a higher TUNEL incidence, and this relationship was also found for total thiol levels. Hence, both overoxidation and a low total number of thiol groups predestine for sperm apoptosis. CONCLUSIONS: Chromatin characteristics reflecting an incomplete nucleosome to protamine remodelling were found in subfertile males. Sperm apoptosis is related to both incomplete remodelling and protamine overoxidation.
Sammy Boussiba - One of the best experts on this subject based on the ideXlab platform.
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chromochloris zofingiensis chlorophyceae divides by consecutive multiple fission cell cycle under batch and continuous cultivation
Biology, 2021Co-Authors: Idan Koren, Sammy Boussiba, Inna Khozingoldberg, Aliza ZarkaAbstract:Several green algae can divide by multiple fission and spontaneously synchronize their cell cycle with the available light regime. The yields that can be obtained from a microalgal culture are directly affected by cell cycle events. Chromochloris zofingiensis is considered as one of the most promising microalgae for biotechnological applications due to its fast growth and the flexible trophic capabilities. It is intensively investigated in the context of bio-commodities production (carotenoids, storage lipids); however, the pattern of cell-cycle events under common cultivation strategies was not yet characterized for C. zofingiensis. In this study, we have employed fluorescence microscopy to characterize the basic cell-cycle dynamics under batch and continuous modes of phototrophic C. zofingiensis cultivation. Staining with SYBR green—applied in DMSO solution—enabled, for the first time, the clear and simple visualization of polyNuclear stages in this microalga. Accordingly, we concluded that C. zofingiensis divides by a consecutive pattern of multiple fission, whereby it spontaneously synchronizes growth and cell division according to the available illumination regime. In high-light continuous culture or low-light batch culture, C. zofingiensis cell-cycle was completed within several light-dark (L/D) cycles (14 h/10 h); however, cell divisions were synchronized with the dark periods only in the high-light continuous culture. In both modes of cultivation, daughter cell release was mainly facilitated by division of 8 and 16-polyNuclear cells. The results of this study are of both fundamental and applied science significance and are also important for the development of an efficient Nuclear Transformation system for C. zofingiensis.
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Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales)
Algal Research, 2015Co-Authors: Revital Sharon-gojman, Edo Maimon, Stefan Leu, Aliza Zarka, Sammy BoussibaAbstract:An advanced shuttle-vector for efficient Nuclear Transformation and genetic engineering of Haematococcus pluvialis has been developed and tested by inserting linked trans-genes. The phytoene desaturase (pds) gene mutated in the codon for the amino acid at position 504 (from leucine to arginine), yields an enzyme resistant to the herbicide norflurazon and is used as an endogenous selection marker for Transformation of H. pluvialis. A novel cloning vector allowing insertion of additional genes, both at the 5' and the 3' end of the mutated pds, has been designed by inserting the selection marker into the cloning vector pBluescript II SK(-) to give pBS-pds featuring the genomic mutated pds including 2000bp of its promoter. In a second version pBS-pds short was created, by shortening the promoter sequence to 1000bp. Unique restriction sites 5' and 3' of the selection marker have been reserved for insertion and simultaneous Transformation with two transgenes. Transformation efficiency was significantly better than previously reported, achieving Transformation frequencies of 2×10-5 both with long and short promoters, as well as with linear constructs. An expression cassette for the ble derived from vector pGenD-ble was inserted into pBS-pds either 5' or 3' of the pds, and successfully transformed into H. pluvialis, resulting in engineered strains weakly expressing the ble mRNA driven by the Chlamydomonas reinhardtii PsaD promoter. Both circular plasmids, as well as linear DNA fragments only consisting of the ble cassette fused to the pds selection marker were used successfully to engineer H. pluvialis. The plasmid constructs presented here, as well as the use of an endogenous dominant selection marker, represent a blueprint for the future successful production of safe, genetically modified microalgae, for the possible production of high value products or biofuels.
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Development of a Nuclear Transformation system for oleaginous green alga Lobosphaera (Parietochloris) incisa and genetic complementation of a mutant strain, deficient in arachidonic acid biosynthesis
PLoS ONE, 2014Co-Authors: Boris Zorin, Omer Grundman, Nicolas Tourasse, Stefan Leu, Yuval Kaye, Olivier Vallon, Michal Shapira, Inna Khozin-goldberg, Sammy BoussibaAbstract:Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2-5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in Transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful Transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.
