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At least 199 records · Page 11Linked to original sources
PubMed1999

Aminoacylation of tRNAs encoded by Chlorella virus CVK2.

Viruses that infect certain strains of the unicellular green alga, Chlorella, have a large, linear dsDNA genome that is 330-380 kb in size; this genomic size is the largest known among viruses and is equivalent to approximately 60% of the smallest prokaryotic genome of Mycoplasma genitalium (580 kb). Besides many putative protein-coding genes, a cluster of 10-15 tRNA genes is present in these viral genomes. Some of these tRNA genes contain peculiar insertions. In infected host cells, the viral tRNAs of CVK2, a Chlorella virus isolate, have been demonstrated to be cotranscribed as a large precursor, approximately 1.0 kb in size, that is precisely processed into individual mature tRNA species. Acidic Northern blot analysis of eight of these tRNAs has revealed that they are actually aminoacylated in vivo, indicating their involvement in viral protein synthesis. They may help the virus reach maximal replication potential by overcoming codon usage barriers that exist between the virus and its host. These results provide evidence that some components of the host protein synthesis machinery can be replaced by viral gene products. This is the first report of tRNA aminoacylation encoded by viruses of eukaryotes.

Acylation↗
PubMed1999

Characterization of two chitinase genes and one chitosanase gene encoded by Chlorella virus PBCV-1.

Chlorella virus PBCV-1 encodes two putative chitinase genes, a181/182r and a260r, and one chitosanase gene, a292l. The three genes were cloned and expressed in Escherichia coli. The recombinant A181/182R protein has endochitinase activity, recombinant A260R has both endochitinase and exochitinase activities, and recombinant A292L has chitosanase activity. Transcription of a181/182r, a260r, and a292l genes begins at 30, 60, and 60 min p.i., respectively; transcription of all three genes continues until the cells lyse. A181/182R, A260R, and A292L proteins are first detected by Western blots at 60, 90, and 120 min p.i., respectively. Therefore, a181/182r is an early gene and a260r and a292l are late genes. All three genes are widespread in chlorella viruses. Phylogenetic analyses indicate that the ancestral condition of the a181/182r gene arose from the most recent common ancestor of a gene found in tobacco, whereas the genealogical position of the a260r gene could not be unambiguously resolved.

Amino Acid Sequence↗
PubMed2000

Characterization of a beta-1,3-glucanase encoded by chlorella virus PBCV-1.

Sequence analysis of the 330-kb chlorella virus PBCV-1 genome revealed an open-reading frame, A94L, that encodes a protein with significant amino acid identity to Glycoside Hydrolase Family 16 beta-1,3-glucanases. The a94l gene was cloned and the protein was expressed as a GST-A94L fusion protein in Escherichia coli. The recombinant A94L protein hydrolyzed the beta-1,3-glucose polymer laminarin and had slightly less hydrolytic activity on beta-1,3-1, 4-glucose polymers, lichenan and barley beta-glucan. The recombinant enzyme had the highest activity at 65 degrees C and pH 8. We predicted that the a94l-encoded beta-1,3-glucanase is involved in degrading the host cell wall either during virus release and/or is packaged in the virion particle and involved in virus entry. Therefore, we expected a94l to be expressed late in virus infection. However, contrary to expectations, both the a94l mRNA and the A94L protein appeared 15 min after PBCV-1 infection and disappeared 60- and 120-min p.i. postinfection, respectively, indicating that a94l is an early gene. Twenty-seven of 42 chlorella viruses contained the a94l gene. To our knowledge, this is the first report of a virus-encoded beta-1,3-glucanase.

Amino Acid Sequence↗
PubMed2001

Intron conservation in the DNA polymerase gene encoded by Chlorella viruses.

Previously we reported that 19 of 42 viruses that infect Chlorella strain NC64A (NC64A viruses) contain a short, nuclear-located, spliceosomal-processed intron in a pyrimidine dimer-specific glycosylase/apyrimidine lyase (pdg) gene. Surprisingly, the nucleotide sequence of the intron region is more conserved than the exon regions of the gene (L. Sun et al., 2000, J. Mol. Evol. 50, 82-92). For comparative purposes, we determined the nucleotide sequence of a similar intron type and its flanking coding regions in the DNA polymerase (dnapol) gene from the same 42 NC64A viruses and also 5 viruses that infect Chlorella strain Pbi. Thirty-eight of the 42 NC64A viruses contained a 101-nucleotide intron and the remaining 4 had an 86-nucleotide intron located in the same position in dnapol. The 4 viruses with the smaller intron in dnapol also have a smaller intron in their pdg gene. There was no intron in the dnapol gene of the 5 Pbi viruses. Phylogenetic analyses indicate that the dnapol genes containing the 86-nucleotide intron represent the ancestral condition among the NC64A viruses. The intron in the dnapol gene is phase 0 (keeps codons intact), which differs from the phase 1 intron in the pdg gene. The intron in the dnapol gene, unlike the pdg intron, was conserved (83 to 100% identical) to about the same extent as the coding regions of the gene (78 to 100% identical).

Amino Acid Sequence↗
PubMed2002

Chitin synthesis in chlorovirus CVK2-infected chlorella cells.

Hyaluronan synthesis in chlorovirus PBCV-1-infected Chlorella cells was previously reported (DeAngelis et al., 1997). In contrast, we report here on the detection, characterization, and expression of a gene for chitin synthase (chs) encoded by chlorovirus CVK2 isolated in Kyoto, Japan. The CVK2 chs gene encoding an open reading frame of 516 aa was expressed as early as 10 min postinfection (p.i.), peaked at 20-40 min p.i., and disappeared at 120-180 min p.i. The chitin polysaccharide began to accumulate as chitinase-sensitive, hair-like fibers on the outside of the virus-infected Chlorella cell wall by 30 min p.i. All chloroviruses without the gene for hyaluronan synthase (has) alternatively contained the chs gene, suggesting the importance of polysaccharide production in the course of virus infection. A few chloroviruses possessed both the chs and has genes and produced chitin and hyaluronan simultaneously. Polysaccharide accumulation on the algal surface may protect virus-infected algae from uptake by other organisms, such as protozoa. Since CVK2 was reported to encode two chitinases and one chitosanase, CVK2 is a very peculiar virus that encodes enzymes required for both the synthesis and the degradation of chitin materials.

Amino Acid Sequence↗
PubMed2002

Ornithine decarboxylase encoded by chlorella virus PBCV-1.

Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A207R, which encodes a protein with 37-41% amino acid identity to ornithine decarboxylase (ODC) from many eukaryotic organisms. The a207r gene was cloned and the protein was expressed as a His-A207R fusion protein in Escherichia coli. The recombinant protein catalyzes pyridoxal 5'-phosphate-dependent decarboxylation of ornithine to putrescine, the first step in the polyamine biosynthetic pathway. The enzyme has a pH optimum of 9.0 and a temperature optimum of 42 degrees C, and it requires dithiothreitol for maximal activity. The enzyme has a K(m) for ornithine of 0.78 mM and a specific activity of 100 micromol/min/mg protein. PBCV-1 ODC is quite sensitive to the competitive inhibitor L-arginine and the irreversible inhibitor difluoromethylarginine but it is less sensitive to the irreversible inhibitor difluoromethylornithine. The a207r gene is expressed both early and late in PBCV-1 infection and is highly conserved among the chlorella viruses. The 42-kDa PBCV-1 ODC (372 amino acids) is the smallest ODC in the databases and, to our knowledge, is the first virus-encoded ODC.

Amino Acid Sequence↗
PubMed1994

Effect of nickel on certain physiological and biochemical behaviors of an acid tolerant Chlorella vulgaris.

This study concerns the inhibitory effects of acid pH and nickel on growth, nutrient (NO3- and NH4+) uptake, carbon fixation, O2 evolution, electron transport chain and enzyme (nitrate reductase and ATPase) activities of acid tolerant and wild-type strains of Chlorella vulgaris. Though a general reduction in all these variables was noticed with decreasing pH, the tolerant strain was found to be metabolically more active than the wild-type. A reduced cation (NH4+, Na+, K+ and Ca2+) uptake, coupled with a facilitated influx of anions (NH4+, PO4(3-) and HCO3-), suggested the development of a positive membrane potential in acid tolerant Chlorella. Nevertheless, a tremendous increase in ATPase activity at decreasing pH revealed the involvement of superactive ATPase in exporting H+ ions and keeping the internal pH neutral. A difference in Na+ and K+ efflux of the two strains at decreasing pH suggests there is a difference in membrane permeability. The low toxicity of Ni in the acid tolerant strain may be due to the low Ni uptake brought about by a change in membrane potential as well as in permeability. Hence, the development of superactive ATPase and a change in both membrane potential and permeability not only offers protection against acidity, but also co-tolerance to metals.

Adenosine Triphosphatases↗
PubMed1978

The role of vanadium in green plants. III. Influence on cell division of Chlorella.

Vanadium, although essential for growth and chlorophyll formation in unicellular green algae, reveals toxic influences on cell division of Chlorella pyrenoidosa, these disturbances arising in the same range of V-concentrations as the known positive effects of the trace metal. In permanent light, as documented by cell volume statistics, vanadium (4-10(-7) g-at/1 as NH4VO3) causes a significant shift of the distribution maxima to higher values of the algal cell volume, the shift having its optimum at 10(-5) g-at V/1. It is documented in pH-constant liquid culture that this effect is not due to a change of pH in the nutrient medium. Under synchronous conditions of algal cultivation (16:8h), vanadium causes a total arrest of cell division after 3 periods; this stop lasts for the next 3 cycles. Afterwards, asynchronous divisions newly occur and lead to generally larger autospores. Staining of algal cell nuclei revealed an inhibitory V-effect on nuclear division, yielding giant nuclei with multiple sets of chromosomes, and thereby limiting cell division. Under these conditions, Chlorella pyrenoidosa is not synchronizable in presence of vanadium.

Cell Division↗
PubMed1976

[On the essential oil of green algae. III. The oils of some Chlorella mutants (author's transl)].

The stability against mutation of essential oil formation in the genus Chlorella is similar to that of hydrogenase. Both characters are therefore of special value in Chlorella taxonomy. All the 21 mutants of C. fusca C-1.1.10 produce essential oils, but only a few of them produce proazulenes. Two mutants of C. kessleri C-1.1.12 produce essential oils and proazulenes like the wild type.

Chlorella↗
PubMed1978

The cryopreservation of Chlorella. 4. Accumulation of lipid as a protective factor.

Following growth under sub-optimal concentrations of nutrients, cells of Chlorella emersonii accumulated lipid and became more resistant to the damage caused by freezing and thawing. These results suggest that the factor responsible for the cold hardening of some Chlorella spp is not the effect of low temperatures per se but simply that of the reduced metabolic rate. Evidence is given that the post-thaw injury observed following rapid rates of cooling is associated with the vacuole.

Chlorella↗
PubMed1976

Content of fatty acids of Chlorella kessleri in a deep tank fermentation.

Chlorella kessleri cultivated in a deep tank contained 4.8% of non-polar lipid; 51% of this fraction represents saturated fatty acids, 7% unsaturated fatty acids. Our investigation of the fatty acids profile demonstrated even- and odd-numbered saturated and unsaturated fatty acids ranging from C12 to C20. Unlike in other Chlorella species, stearic acid was the dominant fatty acid found. Also shown was an elevated C16:0 fatty acid content and a reduced level of unsaturated fatty acids.

Chlorella↗
PubMed1999

Utilization of xylose for growth by the eukaryotic alga, Chlorella.

A green alga, Chlorella, was found to be capable of utilizing xylose or other pentose sugars (xylitol, arabinose) for enhanced growth rates when grown in the light, but not when grown heterotrophically in the dark. With selection for growth in xylose-containing medium, it was possible to improve dramatically the ability of selected Chlorella strains to grow on xylose mixotrophically. Growth on arabinose or xylitol was not changed in the xylose-selected strains.

Arabinose↗
PubMed2006

Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation.

A eukaryotic microalga, Chlorella sp. DT, was transformed with the Bacillus megaterium strain MB1 merA gene, encoding mercuric reductase (MerA), which mediates the reduction of Hg2+ to volatile elemental Hg0. The transformed Chlorella cells were selected first by hygromycin B and then by HgCl2. The existence of merA gene in the genomic DNA of transgenic strains was shown by polymerase chain reaction amplification, while the stable integration of merA into genomic DNA of transgenic strains was confirmed by Southern blot analysis. The ability to remove Hg2+ in merA transgenic strains was higher than that in the wild type. The merA transgenic strains showed higher growth rate and photosynthetic activity than the wild type did in the presence of a toxic concentration of Hg2+. Cultured with Hg2+, the expression level of superoxide dismutase in transgenic strains was lower than that in the wild type, suggesting that the transgenic strains faced a lower level of oxidative stress. All the results indicated that merA gene was successfully integrated into the genome of transgenic strains and functionally expressed to promote the removal of Hg2+.

Bacillus megaterium↗
PubMed2006

Purification and characterization of cadmium-binding protein from unicelluar alga Chlorella sorokinian.

The unicellular green alga Chlorella sorokiniana ANA9 is highly resistant to heavy metals, and its metal-binding proteins are induced in the presence of cadmium. A novel cadmium-binding protein in C. sorokiniana cultured in 100 mg/l cadmium ions for 4 days was isolated and characterized. The crude protein extract was obtained by cell disruption and partly purified by ammonium sulfate precipitation. After purification by anion-exchange chromatography with diethylaminoethyl (DEAE)-Sepharose CL-6B, the protein was further purified by gel filtration with Sephacryl S-100, followed by Sephadex G-75. The molecular weight of the purified protein was determined to be 11.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cadmium binding capacity of the purified protein was 119 microg/mg. The involvement of thiol coordination in metal-ion binding was confirmed by measuring the ultraviolet spectrum. This article is the first to describe the metallothionein-like cadmium-binding protein from Chlorella species, the expression of which is induced by cadmium exposure.

Chlorella↗
PubMed2001

Transcriptional regulation of the nitrate reductase gene in Chlorella vulgaris: identification of regulatory elements controlling expression.

Nitrate reductase (NR), the rate-limiting and primary control point of the nitrate assimilation pathway, is regulated at transcriptional and post-transcriptional levels. To better understand how NR is regulated at the transcriptional level in Chlorella vulgaris, studies were performed to identify the factors regulating NR expression. Sequence analysis of the NR promoter identified a number of potential sites that were investigated by mobility shift assays. Of the protein-binding sites found, several., such as USF and E2F are likely involved in the basal NR gene transcription. An indirect repeat sequence with similarity to the sequence recognized by the common plant regulatory factor was identified and shown to bind a Chlorella protein in vitro. Mobility shift assays of a consensus GATA element indicated that proteins able to specifically bind this element are constitutive, regardless of the nitrogen source. Mutational analysis revealed that the GATA core is required for protein binding in vitro. Additionally, a NIT2 zinc-finger domain/glutathione S-transferase fusion protein was found to bind in a sequence-specific manner to this site. In Neurospora crassa and Aspergillus nidulans, consensus GATA elements are bound by the NIT2 protein, which has a major role in the expression of nitrogen-metabolizing genes. The ability of the GATA element to function as a nitrogen response element (NRE) was further examined by in vivo foot-printing. The protection of guanines in the GATA core and surrounding region was observed only in cells grown in the presence of nitrate. These data confirm that a single GATA element has a role in regulating the expression of NR in C. vulgaris.

Binding Sites↗
PubMed2002

The stability of the Chlorella nitrate reductase mRNA is determined by the secondary structure of the 5'-UTR: implications for posttranscriptional regulation of nitrate reductase.

Nitrate reductase (NR; EC 1.6.1.1-3) can be controlled at both transcriptional and posttranscriptional levels. Here we describe stability of NR mRNA as a mechanism of control. The NR gene in Chlorella vulgaris (Warburg strain) transcribes a stable mRNA and an unstable mRNA. In-vitro-synthesized transcripts representing these mRNAs show the same stability characteristics. The unstable mRNA is 30 nucleotides longer at the 5'-UTR compared to the stable mRNA. Using an RNA-folding program the 5'-UTR of the longer unstable RNA showed a more extensive stem-loop structure compared to the more linear form of the shorter stable mRNA. Transcripts representing RNAs with intermediate 5'-UTRs folded similarly to the long form and were unstable, or similarly to the short form and were more stable. Thus the secondary structure of the 5'-UTR of NR mRNA is important in the stability of NR transcripts in Chlorella and allows the cell to respond to changes in nitrogen source in an energy-efficient manner.

5' Untranslated Regions↗
PubMed2005

Glucose-6P dehydrogenase in Chlorella sorokiniana (211/8k): an enzyme with unusual characteristics.

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki(NADPH) is 103 microM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki(NADPH) decreased to 48 microM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.

Chlorella↗