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1.  New restriction endonuclease CviRI cleaves DNA at TG/CA sequences. 
Nucleic Acids Research  1994;22(19):3928-3929.
A new type II restriction endonuclease, CviRI, was isolated from virus XZ-6E infected chlorella cells. CviRI is the first restriction endonuclease to recognize the sequence 5'-TGCA-3' and cleaves DNA between the G and C residues to produce blunt-end termini. Methylation of the adenine or cytosine in 5'-TGCA-3' sequences prevents CviRI cleavage. Due to its sequence specificity, CviRI may be especially useful for detecting mutant alleles of many heritable human genetic diseases.
PMCID: PMC308391  PMID: 7937114
2.  The Phycodnaviridae: The Story of How Tiny Giants Rule the World 
The family Phycodnaviridae encompasses a diverse and rapidly expanding collection of large icosahedral, dsDNA viruses that infect algae. These lytic and lysogenic viruses have genomes ranging from 160 to 560 kb. The family consists of six genera based initially on host range and supported by sequence comparisons. The family is monophyletic with branches for each genus, but the phycodnaviruses have evolutionary roots that connect them with several other families of large DNA viruses, referred to as the nucleocytoplasmic large DNA viruses (NCLDV).
PMCID: PMC2908299  PMID: 19216434
3.  Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC. 
Nucleic Acids Research  1998;26(17):3961-3966.
A novel gene encoding a cytosine-5-DNA methyltransferase recognizing the dinucleotide GpC was cloned from Chlorella virus NYs-1 and expressed in both Escherichia coli and Saccharomyces cerevisiae . The gene was sequenced and a predicted polypeptide of 362 amino acids with a molecular weight of 41.903 kDa was identified. The protein contains several amino acid motifs with high similarity to those of other known 5-methylcytosine-forming methyltransferases. In addition, this enzyme, named M. Cvi PI, shares 66% identity and 76% similarity with M. Cvi JI, the only other cytosine-5-DNA methyltransferase cloned from a Chlorella virus. The short, frequently occurring recognition sequence of the new methyltransferase will be very useful for in vivo chromatin structure studies in both yeast and higher organisms.
PMCID: PMC147793  PMID: 9705505
4.  Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV. 
The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photodamaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.
PMCID: PMC168447  PMID: 9097450
5.  Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1. 
Journal of Virology  1997;71(3):1931-1937.
We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.
PMCID: PMC191272  PMID: 9032324
6.  Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1. 
Journal of Virology  1996;70(10):6658-6664.
We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus. We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.
PMCID: PMC190707  PMID: 8794301
7.  Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A. 
Nucleic Acids Research  1996;24(13):2463-2469.
R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.
PMCID: PMC145972  PMID: 8692682
9.  Leptospira genomes are modified at 5'-GTAC. 
Journal of Bacteriology  1993;175(12):3913-3915.
Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI (5'-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to RsaI digestion. Modification at 5'-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria.
PMCID: PMC204811  PMID: 8509345
10.  Viruses and viruslike particles of eukaryotic algae. 
Microbiological Reviews  1991;55(4):586-620.
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. These viruses can be produced in large quantities, assayed by plaque formation, and analyzed by standard bacteriophage techniques. The viruses are structurally similar to animal iridoviruses, their genomes are similar to but larger (greater than 300 kbp) than that of poxviruses, and their infection process resembles that of bacteriophages. Some of the viruses have DNAs with low levels of methylated bases, whereas others have DNAs with high concentrations of 5-methylcytosine and N6-methyladenine. Virus-encoded DNA methyltransferases are associated with the methylation and are accompanied by virus-encoded DNA site-specific (restriction) endonucleases. Some of these enzymes have sequence specificities identical to those of known bacterial enzymes, and others have previously unrecognized specificities. A separate rod-shaped RNA-containing algal virus has structural and nucleotide sequence affinities to higher plant viruses. Quite recently, viruses have been associated with rapid changes in marine algal populations. In the next decade we envision the discovery of new algal viruses, clarification of their role in various ecosystems, discovery of commercially useful genes in these viruses, and exploitation of algal virus genetic elements in plant and algal biotechnology.
PMCID: PMC372839  PMID: 1779928
11.  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. They contained numerous structural proteins and large double-stranded DNA genomes of at least 300 kilobase pairs. Each of the DNAs from the Chinese viruses contained 5-methyldeoxycytosine, which varied from 12.6 to 46.7% of the deoxycytosine, and N6-methyldeoxyadenosine, which varied from 2.2 to 28.3% of the deoxyadenosine. Four of the Chinese virus DNAs hybridized extensively with DNA from the American virus PBCV-1, and three hybridized poorly.
PMCID: PMC202831  PMID: 2847652
12.  DNA methyltransferase induced by PBCV-1 virus infection of a Chlorella-like green alga. 
Molecular and Cellular Biology  1986;6(5):1440-1445.
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.
PMCID: PMC367668  PMID: 3537703
13.  Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga. 
Molecular and Cellular Biology  1986;6(5):1430-1439.
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.
PMCID: PMC367667  PMID: 3023890
14.  Synthesis and localization of a development-specific protein in sclerotia of Sclerotinia sclerotiorum. 
Journal of Bacteriology  1985;163(2):696-703.
A development-specific protein (SSP) makes up about 35 to 40% of the total protein in sclerotia of the fungus Sclerotinia sclerotiorum. The protein consists of three charge isomers, with one isomer making up 80 to 90% of the total. In vitro translation of poly(A)+ RNA isolated from cells in early stages of sclerotia formation revealed that 44% of the amino acids incorporated was into SSP. In vivo- and in vitro-synthesized forms of SSP migrated at identical rates on both isoelectric focusing and denaturing polyacrylamide gels, indicating that SSP was not synthesized as a larger precursor. This was significant because SSP accumulated in membrane-bound, organellelike structures which resemble protein bodies found in seeds of many higher plants.
PMCID: PMC219177  PMID: 2410402
15.  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.
PMCID: PMC238550  PMID: 4004247
16.  Biosynthesis and degradation of storage protein in spores of the fungus Botryodiplodia theobromae. 
Journal of Bacteriology  1983;155(2):601-606.
Muiridin, a spore-specific protein of the fungus Botryodiplodia theobromae, comprises about 25% of the mature pycnidiospore protein. It has an apparent molecular weight of 16,000 to 17,000 and is rich in glutamine, asparagine, and arginine. Muiridin is synthesized in developing spores via a precursor with an apparent molecular weight of 24,000. Two other polypeptides present in young developing spores with apparent molecular weights of 18,000 and 15,000 are immunologically related to muiridin. We propose a pathway for muiridin synthesis. Muiridin is actively degraded during the germination of spores from 30-day-old cultures. This degradation is independent of exogenous amino acids in the germination medium. In contrast, glutamine and, to a lesser extent, asparagine partially inhibit the degradation of muiridin during germination of spores from 7-day-old cultures.
PMCID: PMC217728  PMID: 6874639
17.  In vitro translation of the three bacteriophage phi 6 RNAs. 
Journal of Virology  1980;35(1):249-251.
In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9.
PMCID: PMC288801  PMID: 6997510
18.  Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase. 
Journal of Virology  1980;33(2):769-773.
The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.
PMCID: PMC288602  PMID: 7411690
19.  Presence of a major (storage?) protein in dormant spores of the fungus Botryodiplodia theobromae. 
Journal of Bacteriology  1979;138(2):650-652.
Approximately 23% of the protein isolated from dormant spores of Botryodiplodia theobromae consisted of a single polypeptide; the polypeptide is probably degraded during germination.
PMCID: PMC218224  PMID: 438139
20.  Simple procedure for disruption of fungal spores. 
A simple, inexpensive method for disrupting dormant fungal spores is described.
PMCID: PMC242889  PMID: 637553
21.  Absence of spermine in filamentous fungi. 
Journal of Bacteriology  1977;129(1):173-176.
Polyamines were examined in several yeasts and filamentous fungi. Whereas putrescine, spermidine, and spermine were present in the yeasts, spermine was not detected in any of the filamentous fungi.
PMCID: PMC234911  PMID: 830639
22.  Influence of Molecular Weights of Bacteriophage φ6 Double-Stranded Ribonucleic Acids on Interferon Induction 1 
Infection and Immunity  1974;10(1):284-285.
The interferon-inducing capabilities of the three molecular segments of bacteriophage φ6 double-stranded ribonucleic acid increased with increasing molecular weight.
PMCID: PMC414992  PMID: 4842130
23.  Base Composition and Hybridization Studies of the Three Double-Stranded RNA Segments of Bacteriophage φ6 1 
Journal of Virology  1974;13(6):1254-1262.
The three double-stranded ribonucleic acid (dsRNA) segments of the bacteriophage φ6 were isolated and shown to have similar melting temperatures and base compositions. RNA:RNA hybridization experiments with the isolated segments eliminate the possibility that the two smaller dsRNA segments arise from a cleavage of the large dsRNA segment. The two smaller RNA segments reanneal rapidly even at low temperatures; in contrast, the large dsRNA reannealed only at higher temperatures. Evidence is also presented which suggests that the dsRNAs may contain a short single-stranded RNA tail.
PMCID: PMC355445  PMID: 4833610
24.  RNA Polymerase Activity Associated with Bacteriophage φ6 1 
Journal of Virology  1973;12(3):464-471.
The Pseudomonas phaseolicola bacteriophage φ6 incorporated labeled UTP into an acid-insoluble precipitate. Incorporation was dependent on the presence of manganese acetate, ATP, GTP, CTP, and a short heat treatment of the phage; the reaction was stimulated by NH4Cl. The substitution of 14C-ATP, -CTP or -GTP for UTP, together with the appropriate unlabeled ribonucleoside triphosphates, disclosed that CMP was incorporated to the greatest extent followed by GMP, UMP, and AMP. Radioactive RNAs formed by the reaction were resistant to RNases A and T1 in high salt but susceptible to these nucleases in low salt. The labeled RNA co-sedimented and co-electrophoresed with φ6 double-stranded (ds) RNA. However, the distribution of the radioactivity into the three ds-RNA components varied depending on the 14C-ribonucleoside triphosphate used in the reaction. The incorporation of UMP was primarily into the two smaller ds-RNA segments, GMP primarily into the large ds-RNA segment, and CMP and AMP were about equally distributed into all three ds-RNA segments.
PMCID: PMC356652  PMID: 4747635
25.  Bacteriophage φ6: a Lipid-Containing Virus of Pseudomonas phaseolicola1 
Journal of Virology  1973;11(5):799-805.
The purification and properties of a lipid-containing bacteriophage, φ6, are described. The phage contains a lipid envelope which is probably essential for infection. Infectivity of φ6 was lost in the presence of organic solvents, sodium deoxycholate, and phospholipase A. The fatty acid composition of the phage lipid was similar to that of the Pseudomonas phaseolicola host cells. The phage was composed of about 25% lipid, 13% RNA, and 62% protein. The buoyant density of φ6 was 1.27 g/ml in cesium chloride. The morphology of φ6 was unusual; it had a polyhedral head of about 60 nm surrounded by a membranous, compressible envelope which appeared to assume an elongated configuration upon attachment to pili. The adsorption rate constant was 3.3 × 10−10 ml/min in a semi-synthetic medium and 3.8 × 10−10 ml/min in a nutrient broth-yeast extract medium. The latent period was shorter in the former medium (80-115 min compared with 120-160 min), and the average burst size was larger (250-400 compared with 125-150). The eclipse period coincided with the latent period.
PMCID: PMC355178  PMID: 16789137

Results 1-25 (36)