DNA methyltransferase induced by PBCV-1 virus infection of a Chlorella-like green alga.

A DNA methyltransferase was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1. The enzyme recognized the sequence GATC and methylated deoxyadenosine solely in GATC sequences. Host DNA, which contains GATC sequences, but not PBCV-1 DNA, which contains GmATC sequences, was a good substrate for the enzyme in vitro. The DNA methyltransferase activity was first detected about 1 h after viral infection; PBCV-1 DNA synthesis and host DNA degradation also began at about this time. The appearance of the DNA methyltransferase activity required de novo protein synthesis, and the enzyme was probably virus encoded. Methylation of DNAs with the PBCV-1-induced methyltransferase conferred resistance of the DNAs to a PBCV-1-induced restriction endonuclease enzyme described previously (Y. Xia, D. E. Burbank, L. Uher, D. Rabussay, and J. L. Van Etten, Mol. Cell. Biol. 6:1430-1439). We propose that the PBCV-1-induced methyltransferase protects viral DNA from the PBCV-1-induced restriction endonuclease and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.     

The mitochondrial genome of an exsymbiotic Chlorella-like green alga

Mitochondrial (mt) DNA from the unicellular, exsymbiotic Chlorella-like green alga, strain Nla was isolated and cloned. The mtDNA has a buoyant density of 1.692 g/ml in CsCl and an apparent G/C base composition of 32.5%. The genome contains approximately 76 kbp of DNA based on restriction fragment summation and electron microscopic measurements. A map of restriction endonuclease sites using Sst I, Bam I, Sal I and Xho I was generated. The genome maps as a circular molecule and appears as such under the electron microscope. Eight genes were assigned to the map by hybridization to specific restriction fragments using heterologous mt-encoded specific probes. These include the genes for subunits 6, 9, and alpha of the F₀-F₁ ATPase complex, the large and small subunit rRNAs, cytochrome oxidase subunits I and II, and apocytochrome b.     

The chloroplast genome of an exsymbiotic Chlorella-like green alga

Chloroplast DNA was isolated and cloned from Chlorella, strain N1a, exsymbiotic with Paramecium bursaria. BamHI, SalI, SstI, KpnI and XhoI restriction fragments of the DNA were assembled into a circular map. The genome consists of approximately 120 kbp of DNA, has a G/C content of 38%, and contains only a single copy of the rRNA cistron. The rRNA cistron is small, 5000–8000 bp, and the 16S and 23S genes are separated by less than 2000 bp.     

IL-3A virus infection of a Chlorella-like green alga induces a DNA restriction endonuclease with novel sequence specificity.

A type II restriction endonuclease, named CviJI, was isolated from a eukaryotic Chlorella-like green alga infected with the dsDNA containing virus IL-3A. CviJI is the first restriction endonuclease to recognize the sequence PuGCPy; CviJI cleaves DNA between the G and C. Methylation of the cytosine in PuGCPy sequences prevents cleavage by CviJI. CviJI cleaved DNA into smaller but defined fragments in the presence of ATP. This "star" activity was stimulated by dithiothreitol and/or S-adenosylmethionine but did not occur under conditions which favor "star" activity of other restriction endonucleases.     

Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga.

An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.     

Restriction endonuclease activity induced by NC-1A virus infection of a Chlorella-like green alga.

A type II restriction endonuclease, CviBI, was isolated from a eukaryotic, Chlorella-like green alga infected with the dsDNA containing virus NC-1A. The enzyme recognizes the sequence GANTC and cleaves DNA between the G and A. Methylation of deoxyadenosine in the GANTC sequence probably inhibits enzyme activity. In vitro CviBI cleaves host nuclear DNA but not viral DNA. A survey of 18 other viruses which infect the same Chlorella sp. revealed that infection with 5 of these viruses also induced a restriction endonuclease which cleaves DNA into the same size fragments as CviBI.     

A site-specific single strand endonuclease activity induced by NYs-1 virus infection of a Chlorella-like green alga.

A site-specific endonuclease was isolated from a eukaryotic Chlorella-like green alga infected with the dsDNA-containing virus NYs-1. The enzyme recognizes the sequence 5'-CC-3' and cleaves 5' to the first C. It cleaves 5'-CmC-3' sequences but not 5'-mCC-3' sequences. The enzyme creates breaks in dsDNA whenever two 5'-CC-3' sequences on opposite strands are close enough for the two strands to separate; when the 5'-CC-3' sequences on opposite strands are further apart only a portion of the strands separate. Consequently, NYs-1 endonuclease does not produce a completely stable DNA digestion pattern. The enzyme probably does not cleave ssDNA and definitely does not cleave ssRNA or dsRNA.     

Amplification of DNA polymerase gene fragments from viruses infecting microalgae.

Nested PCR with three highly degenerate primers was used for amplification and identification of DNA polymerase (pol) genes from viruses which infect three genera of microalgae. Group-specific primers (AVS1 and AVS2) were designed on the basis of inferred amino acid sequences unique to the DNA pol genes of viruses (PBCV-1 and NY-2A) that infect an endosymbiotic Chlorella-like alga (Chlorophyceae) and a virus (MpV-SP1) which infects the photosynthetic flagellate Micromonas pusilla (Prasinophyceae). In addition, a nested primer (POL) was designed on the basis of the highly conserved amino acid sequence YGDTDS found in most B-family (alpha-like) DNA pol genes. These primers were used to amplify DNA from the three viruses, PBCV-1, NY-2A, and MpV-SP1, for which the primers were designed, as well as eight clonal isolates of genetically distinct viruses which infect M. pusilla and others which infect Chrysochromulina spp. (Prymnesiophyceae), suggesting that these are a group of related viruses. In contrast, no product resulted from using DNA from viruses which infect the marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae), suggesting that these viruses may not be closely related to those that infect microalgae. These primers were also used to amplify DNA from natural virus communities. Our results indicate that nested PCR, even under low-stringency conditions, can be used as a rapid method to verify the presence in seawater of a group of related viruses which infect microalgae. Sequence analysis of these fragments should provide information on the genetic diversity and potentially the phyletic relationships among these viruses.(ABSTRACT TRUNCATED AT 250 WORDS)     

New members of a group of DNA viruses infecting brown algae

Since 1990 virus infections have been described in six brown algal species of the genera Ectocarpus, Feldmannia, Hincksia and Myriotrichia. These pathogens can be experimentally transmitted to healthy isolates of their hosts. A synopsis including new molecular and biochemical data shows that these viruses share common characteristics: genomes of double-stranded DNA, infection mode, morphology, extended temperence, and narrow host-specificity. These properties distinguish the brown algal viruses from all other known plant viruses.     

Characteristics and sequence of phosphoglycolate phosphatase from a eukaryotic green alga Chlamydomonas reinhardtii

Phosphoglycolate phosphatase (PGPase), a key enzyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The enzyme was an approximately 65-kDa homodimer with a pI value of 5.1 composed of approximately 32-kDa subunits not connected by any S-S bridges. It was also highly specific for phosphoglycolate with a K(m) value of 140 microm and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl(2), and it recovered competitively following the addition of MgCl(2) or EGTA. A mobility shift was observed in SDS-polyacrylamide gel electrophoresis by the addition of CaCl(2), indicating that the enzyme binds to Ca(2+). The N-terminal region of amino acid sequence deduced from cDNA sequence that was not contained in the purified PGPase had similar characteristics to those of typical stroma-targeting transit peptides in C. reinhardtii. The following region of the deduced sequence containing 302 amino acid residues was similar to p-nitrophenylphosphatase-like proteins, although the purified PGPase did not hydrolyze p-nitrophenylphosphate. Genomic DNA fragments from wild type containing the sequence homologous to the cDNA for PGPase complemented the PGPase-deficient mutant pgp1. Possible regulatory mechanisms during adaptation to limiting CO(2) were discussed based on the characteristics of the purified PGPase and the deduced amino acid sequence.     

A survey for viruses from fresh water that infect a eucaryotic chlorella-like green alga.

Viruses which formed plaques on lawns of a eucaryotic, chlorella-like green alga were detected in 37% of the 35 freshwater samples surveyed. Virus populations, monitored in seven locations, fluctuated both qualitatively and quantitatively over an 8-month period.     

A survey for viruses from fresh water that infect a eucaryotic chlorella-like green alga

Viruses which formed plaques on lawns of a eucaryotic, chlorella-like green alga were detected in 37% of the 35 freshwater samples surveyed. Virus populations, monitored in seven locations, fluctuated both qualitatively and quantitatively over an 8-month period.     

Changes in DNA composition during cell differentiation in a green alga

In the coenobia-forming green alga, Hydrodictyon reticulatum, changes in DNA composition during growth and differentiation of reproductive cells were followed. A “loss” of DNA per nucleus could be attributed to underreplication of an amplified DNA fraction being part of a (GC)-rich satellite band in CsCl gradients of DNA from young coenobia. This (GC)-rich satellite DNA contains up to 3 % unique DNA sequences intermingled with repetitive DNA. It does not (or nearly not) contain ribosomal DNA. A possible role of this DNA fraction in differentiation is suggested.     

The minimal eukaryotic ribosomal DNA units in the primitive red alga Cyanidioschyzon merolae.

Cyanidioschyzon merolae is a small unicellular red alga that is considered to belong to one of the most deeply branched taxa in the plant kingdom. Its genome size is estimated to be 16.5 Mbp, one of the smallest among free-living eukaryotes. In the nucleus containing this small genome, one nucleolus is clearly observed, but the molecular basis for the intranuclear structure including ribosomal DNA organization is still unclear. We constructed a bacterial artificial chromosome library for C. merolae 10D composed of two subsets with different insert size distributions. The two subsets have average insert sizes of 97 and 48 kb, representing 10.0- and 6.9-fold genome-equivalent coverage of the haploid genome, respectively. For application to whole-genome shotgun sequencing, the termini of each clone were sequenced as sequence-tagged connectors and mapped on the contigs assigned to chromosomes. Screening for rRNA genes by conventional colony hybridization with high-density filter blots and subsequent sequencing revealed that the C. merolae genome contained the smallest number of ribosomal DNA units among all the eukaryotes examined to date. They consist of only 3 single units of rRNA genes distributed on separate chromosomal loci, representing an implication for concerted evolution. Based on these results, the origin and evolution of the nucleolus are discussed.     

A constitutive flavodoxin from a eukaryotic alga

We report the isolation and some properties of a flavodoxin from a eukaryotic organism, the naturally occurring red alga $\text{Chondrus crispus}$. Unlike the situation with most other organisms the flavodoxin, under normal growth conditions, is the predominantly formed low-potential electron carrier, an accompanying ferredoxin occurring in only very small amounts. The flavodoxin is of molecular weight 21000 and one mole of FMN is present per mole of protein. Reduction of the flavoprotein proceeds via a blue flavosemiquinone radical. The flavodoxin is active both in photosynthetic NADP reduction by broken chloroplasts, and in phosphoroclastic cleavage of pyruvate by cell-free extracts of $\text{Clostridium pasteurianum}$.     

Novel anticancer drug curaxin CBL0137 impairs DNA methylation by eukaryotic DNA methyltransferase Dnmt3a

Novel DNA intercalating anticancer drug curaxin CBL0137 significantly inhibited in vitro DNA methylation by eukaryotic DNA methyltransferase Dnmt3a catalytic domain (Dnmt3a-CD) at low micromolar concentrations (IC₅₀ 3–9 µM). CBL0137 reduced the binding affinity of Dnmt3a-CD to its DNA target, causing up to four-fold increase in the K_d of the enzyme/DNA complex. Binding of CBL0137 to Dnmt3a-CD was not observed. The observed decrease in methylation activity of Dnmt3a-CD in the presence of CBL0137 can be explained by curaxin’s ability to intercalate into DNA.