A single amino acid change restores DNA cytosine methyltransferase activity in a cloned chlorella virus pseudogene.

The chlorella virus PBCV-1 contains an open reading frame, named P17-ORF4, which differs by eight amino acids from a DNA cytosine methyltransferase, M.CviJI, encoded by a different chlorella virus IL-3A. Whereas IL-3A expresses M.

DNA sequencing of four bases using three lanes.

A three-lane DNA sequencing strategy is described which is based on redundant binary coding principles from communications theory. Three-lane sequencing is an efficient, accurate, and flexible strategy, suitable for large-scale automated DNA sequencing. Communications theory and algebraic coding principles can also be applied to sequencing other informational macromolecules.

Chlorella virus PBCV-1 replication is not affected by cytoskeletal disruptors.

The majority, if not the entire life cycle, of the large dsDNA-containing algal virus PBCV-1 occurs in localized regions in the cytoplasm. Thirteen drugs that disrupt the cytoskeleton had no effect on PBCV-1 replication at concentrations which inhibited host growth. Therefore, host cytoskeletal elements do not appear to be important in PBCV-1 morphogenesis.

Viruses and viruslike particles of eukaryotic algae.

Until recently there was little interest or information on viruses and viruslike particles of eukaryotic algae. However, this situation is changing. In the past decade many large double-stranded DNA-containing viruses that infect two culturable, unicellular, eukaryotic green algae have been discovered.

The termini of the chlorella virus PBCV-1 genome are identical 2.2-kbp inverted repeats.

The Chlorella virus PBCV-1 genome is a linear nonpermuted 333-kbp dsDNA molecule with covalently closed hairpin termini. The termini (minus the hairpin) are identical inverted repeats of at least 2185 bases after which the sequence diverges. The inverted repeats contain two small potential open reading frames and several direct repeats.

Molecular cloning and characterization of the gene encoding the adenine methyltransferase M.CviRI from Chlorella virus XZ-6E.

The gene encoding the DNA methyltransferase M.CviRI from Chlorella virus XZ-6E was cloned and expressed in Escherichia coli. M.

Cloning and sequencing the cytosine methyltransferase gene M. CviJI from Chlorella virus IL-3A.

The Chlorella virus IL-3A gene encoding the DNA methyltransferase M.CviJI, which methylates the internal cytosine in (G/A)GC(T/C/G) sequences, was cloned and expressed in Escherichia coli. The region containing the M.

5-Azacytidine-resistant mutants of Chlorella virus IL-3A.

Many dsDNA-containing viruses which infect the unicellular, eukaryotic Chlorella-like green alga strain NC64A encode for DNA methyltransferases and DNA site-specific (restriction) endonucleases. We have hypothesized that these endonucleases help degrade host DNA permitting deoxynucleotides to recycle into virus DNA. This hypothesis was tested by isolating deletion mutants of Chlorella virus IL-3A lacking functional genes for the cytosine methyltransferase M.

A phylogenetic analysis of the mycoplasmas: basis for their classification.

Small-subunit rRNA sequences were determined for almost 50 species of mycoplasmas and their walled relatives, providing the basis for a phylogenetic systematic analysis of these organisms. Five groups of mycoplasmas per se were recognized (provisional names are given): the hominis group (which included species such as Mycoplasma hominis, Mycoplasma lipophilum, Mycoplasma pulmonis, and Mycoplasma neurolyticum), the pneumoniae group (which included species such as Mycoplasma pneumoniae and Mycoplasma muris), the spiroplasma group (which included species such as Mycoplasma mycoides, Spiroplasma citri, and Spiroplasma apis), the anaeroplasma group (which encompassed the anaeroplasmas and acholeplasmas), and a group known to contain only the isolated species Asteroleplasma anaerobium. In addition to these five mycoplasma groups, a sixth group of variously named gram-positive, walled organisms (which included lactobacilli, clostridia, and other organisms) was also included in the overall phylogenetic unit.

Mutual exclusion occurs in a Chlorella-like green alga inoculated with two viruses.

Progeny viruses resulting from dual inoculations with different and near-isogenic viruses of a Chlorella-like green alga were distinguished by immunoblotting. Plaques arising from single cells inoculated with two viruses usually contained only one of the viruses. Thus the viruses mutually exclude one another.

Chlorella viruses contain linear nonpermuted double-stranded DNA genomes with covalently closed hairpin ends.

Pulsed field electrophoresis established that Chlorella viruses contain linear, nonpermuted, 330- to 380-kb dsDNA genomes. Terminal DNA restriction fragments of one virus, PBCV-1, were identified by Bal31 exonuclease digestion; the termini probably contain covalently closed hairpin ends. The end fragments cross-hybridize indicating terminal repetition; the region of repetition extends no more than 2.

Characterization of DNA polymerases in an uninfected and virus PBCV-1-infected green alga--Chlorella strain NC64A.

Chlorella NC64A cells infected with the large double-stranded DNA-containing virus PBCV-1 and uninfected cells were assayed for DNA polymerase activity. Both uninfected and infected cells contained three forms of DNA polymerase activity: (i) an exogenous DNA-dependent 10,000 g soluble fraction, (ii) an exogenous DNA-dependent 10,000 g particulate fraction, and (iii) a DNA-independent 10,000 g particulate fraction. The three DNA polymerase activities in the infected and uninfected cells were distinguished from one another by the conditions required for optimum activity and by their sensitivity to inhibitors.

The amino acid sequence of the eukaryotic DNA [N6-adenine]methyltransferase, M.CviBIII, has regions of similarity with the prokaryotic isoschizomer M.TaqI and other DNA [N6-adenine] methyltransferases.

The sequences of the genes coding for M.CviBIII (from virus NC-1A which infects a eukaryotic alga) [Narva et al., Nucleic Acids Res.

A comparison of viruses infecting two different Chlorella-like green algae.

Five plaque-forming viruses (Pbi viruses) of the unicellular, eukaryotic, exsymbiotic Chlorella-like green alga strain Pbi were isolated from fresh water collected in Germany. The viruses were compared to two previously characterized plaque-forming viruses (NC64A viruses) of Chlorella strain NC64A. The Pbi viruses do not infect Chlorella NC64A and vice versa.

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.

Chlorella viruses isolated in China.

Plaque-forming viruses of the unicellular, eucaryotic, exsymbiotic, Chlorella-like green algae strain NC64A, which are common in the United States, were also present in fresh water collected in the People's Republic of China. Seven of the Chinese viruses were examined in detail and compared with the Chlorella viruses previously isolated in the United States. Like the American viruses, the Chinese viruses were large polyhedra and sensitive to chloroform.

Properties of the Chlorella receptor for the virus PBCV-1.

The virus PBCV-1 attached rapidly, specifically, and irreversibly to the external surface of cell walls of its host, a unicellular, eukaryotic Chlorella-like green alga. Attachment was pH and salt dependent. Each cell contained at least 5 X 10(4) PBCV-1 binding sites and Scatchard analysis indicated that each cell could adsorb 5000 PBCV-1 particles.

Molecular cloning and characterization of the gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A.

The gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A was cloned and expressed in E. coli plasmid pUC8.

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.

Restriction site map of the Chlorella virus PBCV-1 genome.

The virus PBCV-1, which replicates in a Chlorella-like green alga, has a dsDNA genome. The DNA was mapped for BamHI, HindIII, and PstI restriction sites. The resulting map has a size of 333 kbp and is circular-indicating either covalently closed circular DNA or circularly permuted linear DNA.

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.

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.