Genetic variation of chlorella viruses: variable regions localized on the CVK2 genomic DNA.
A physical map of the Chlorella virus CVK2 genomic DNA has been constructed based on a cosmid contig covering the entire genomic region. By using Southern blot analysis with 22 gene probes, the gene arrangement along the genome was compared between CVK2 and PBCV-1, the prototypic member of Phycodnaviridae, whose genomic sequence is now available. The major rearrangements were (1) an insertion of a 20-kbp region around the left end of CVK2 DNA, (2) a duplication of the gene for major capsid protein in CVK2 DNA, (3) deletions/insertions of some open reading frames, and (4) divergence in the terminal inverted repeat sequences. Despite these changes, extensive colinearity was revealed between most of the genes along the CVK2 and PBCV-1 genomes. These data imply that the Chlorella virus genome has an overall high degree of genomic stability, encompassing specific islands of rearrangements. (+info)
The catalytic mechanism of a pyrimidine dimer-specific glycosylase (pdg)/abasic lyase, Chlorella virus-pdg.
The repair of UV light-induced cyclobutane pyrimidine dimers can proceed via the base excision repair pathway, in which the initial step is catalyzed by DNA glycosylase/abasic (AP) lyases. The prototypical enzyme studied for this pathway is endonuclease V from the bacteriophage T4 (T4 bacteriophage pyrimidine dimer glycosylase (T4-pdg)). The first homologue for T4-pdg has been found in a strain of Chlorella virus (strain Paramecium bursaria Chlorella virus-1), which contains a gene that predicts an amino acid sequence homology of 41% with T4-pdg. Because both the structure and critical catalytic residues are known for T4-pdg, homology modeling of the Chlorella virus pyrimidine dimer glycosylase (cv-pdg) predicted that a conserved glutamic acid residue (Glu-23) would be important for catalysis at pyrimidine dimers and abasic sites. Site-directed mutations were constructed at Glu-23 to assess the necessity of a negatively charged residue at that position (Gln-23) and the importance of the length of the negatively charged side chain (Asp-23). E23Q lost glycosylase activity completely but retained low levels of AP lyase activity. In contrast, E23D retained near wild type glycosylase and AP lyase activities on cis-syn dimers but completely lost its activity on the trans-syn II dimer, which is very efficiently cleaved by the wild type cv-pdg. As has been shown for other glyscosylases, the wild type cv-pdg catalyzes the cleavage at dimers or AP sites via formation of an imino intermediate, as evidenced by the ability of the enzyme to be covalently trapped on substrate DNA when the reactions are carried out in the presence of a strong reducing agent; in contrast, E23D was very poorly trapped on cis-syn dimers but was readily trapped on DNA containing AP sites. It is proposed that Glu-23 protonates the sugar ring, so that the imino intermediate can be formed. (+info)
Hyaluronan synthesis in virus PBCV-1-infected chlorella-like green algae.
We previously reported that the chlorella virus PBCV-1 genome encodes an authentic, membrane-associated glycosyltransferase, hyaluronan synthase (HAS). Hyaluronan, a linear polysaccharide chain composed of alternating beta1,4-glucuronic acid and beta1, 3-N-acetylglucosamine groups, is present in vertebrates as well as a few pathogenic bacteria. Studies of infected cells show that the transcription of the PBCV-1 has gene begins within 10 min of virus infection and ends at 60-90 min postinfection. The hyaluronan polysaccharide begins to accumulate as hyaluronan-lyase sensitive, hair-like fibers on the outside of the chlorella cell wall by 15-30 min postinfection; by 240 min postinfection, the infected cells are coated with a dense fibrous network. This hyaluronan slightly reduces attachment of a second chlorella virus to the infected algae. An analysis of 41 additional chlorella viruses indicates that many, but not all, produce hyaluronan during infection. (+info)
Several genes in Chlorella virus strain CVG-1 encode putative virion components.
We have started to characterize the capsid components of European Chlorella virus isolate CVG-1, a member of the Pbi subgroup of the Phycodnaviridae. The major coat protein, Vp49, was biochemically characterized and the amino acid sequence of the N terminus was determined. Subsequently, the corresponding gene was isolated from CVG-1 genomic DNA. Sequence data were compared to those available from PBCV-1 and other Chlorella virus isolates representing the NC64A subgroup of the Phycodnaviridae. The major coat proteins of all strains are homologous and similar in size, but apparently differ in their degree of glycosylation. Like PBCV-1, the major coat protein of CVG-1 is part of a gene family, as two open reading frames with high similarity to Vp49 were also isolated and characterized in this study. The predicted amino acid sequences of the CVG-1 and PBCV-1 virus genes examined show, with one exception, a divergence of about 25%. Taking into account that corresponding genes of NC64A viruses are almost identical, this divergence supports the original placement of the NC64A and Pbi viruses into separate subgroups of the Phycodnaviridae. (+info)
Asymmetric reduction of ethyl 2-methyl 3-oxobutanoate by Chlorella.
Chlorella pyrenoidosa Chick reduced ethyl 2-methyl 3-oxobutanoate to the corresponding alcohols with the diastereomer (anti/syn) ratio of 53/47. The enantiomer excesses of anti-(2S, 3S)- and syn-(2S, 3R)-hydroxy esters were 89 and > 99ee% respectively. C. vulgaris and C. regularis afforded predominantly the syn-isomer, contrary to C. pyrenoidosa. The differences in the activity of reducing ethyl 2-methyl 3-oxobutanoate were observed among three strains of Chlorella. Addition of 2% metal salts slightly increased the chemical yield of the hydroxy ester. (+info)
Expression of a chitinase gene and lysis of the host cell wall during Chlorella virus CVK2 infection.
A chitinase gene (vChti-1) encoded by the Chlorella virus CVK2 was cloned and characterized. The vChti-1 open reading frame consisted of 2508 bp corresponding to 836 amino acid residues. The predicted amino acid sequence contained two sets of a family 18 catalytic domain that is responsible for chitinase activity. Northern blot analysis revealed that the vChti-1 gene was expressed in virus-infected Chlorella cells late in infection, when a single transcript of about 2.5 kb appeared at 120 min postinfection. This result was confirmed by Western blotting with a specific anti-vChti-1 protein antibody; a protein of about 94 kDa was detected specifically beginning at 240 min postinfection and was present until cell lysis. The protein was not incorporated into viral particles but remained in the medium after cell lysis. The vChti-1 protein produced in virus-infected cells showed chitinase activity on zymogram assays. (+info)
Chlorella accelerates dioxin excretion in rats.
We investigated the effects of Chlorella on fecal excretion of polychlorinated dibenzo-p-dioxin (PCDD) congeners and polychlorinated dibenzofuran (PCDF) congeners in Wistar rats administered the rice oil that caused Yusho disease, as a substitute for purified dioxin. The rats were fed 4 g of a control diet or a 10% Chlorella diet containing 0.2 mL of the rice oil once during the 5-d experimental period. The amounts of PCDD and PCDF congeners excreted in feces from d 1 to 5 in the group fed 10% Chlorella were 0.2-11.3 and 0.3-12.8 times greater (P < 0.05), respectively, than those of the control group. We then investigated the fecal excretion of PCDD and PCDF congeners from d 8 to 35 in rats administered 0.5 mL of the rice oil. Rats consumed the basal diet for 1 wk. After 1 wk, they consumed either the basal diet or the 10% Chorella diet. The fecal excretions of PCDD and PCDF congeners in the group fed 10% Chlorella were 0.3-3.4 and 0.5-2.5 times greater (most, P < 0.05), respectively, than those of the control group. Thus, the fecal excretions of PCDD and PCDF congeners were greater in rats fed Chlorella. These findings suggest that the administration of Chlorella may be useful in preventing gastrointestinal absorption and for promoting the excretion of dioxin already absorbed into tissues. Moreover, these findings suggest that Chlorella might be useful in the treatment of humans exposed to dioxin. (+info)
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. (+info)