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D E Burbank

Publications and source records attributed to D E Burbank.

At least 19 recordsLinked to original sources

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↗

Topoisomerase II from Chlorella virus PBCV-1. Characterization of the smallest known type II topoisomerase.

Type II topoisomerases, a family of enzymes that govern topological DNA interconversions, are essential to many cellular processes in eukaryotic organisms. Because no data are available about the functions of these enzymes in the replication of viruses that infect eukaryotic hosts, this led us to express and characterize the first topoisomerase II encoded by one of such viruses. Paramecium bursaria chlorella virus 1 (PBCV-1) infects certain chlorella-like green algae and encodes a 120-kDa protein with a similarity to type II topoisomerases. This protein was expressed in Saccharomyces cerevisiae and was highly active in relaxation of both negatively and positively supercoiled plasmid DNA, catenation of plasmid DNA, and decatenation of kinetoplast DNA networks. Its optimal activity was determined, and the omission of Mg(2+) or its replacement with other divalent cations abolished DNA relaxation. All activities of the recombinant enzyme were ATP dependent. Increasing salt concentrations shifted DNA relaxation from a normally processive mechanism to a distributive mode. Thus, even though the PBCV-1 enzyme is considerably smaller than other eukaryotic topoisomerase II enzymes (whose molecular masses are typically 160-180 kDa), it displays all the catalytic properties expected for a type II topoisomerase.

Chlorella↗

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.

Cell Wall↗

Chlorella virus PBCV-1 encodes functional glutamine: fructose-6-phosphate amidotransferase and UDP-glucose dehydrogenase enzymes.

DNA sequence analysis of the 330-kb Chlorella virus PBCV-1 genome unexpectedly revealed several open reading frames which encode proteins that are homologous to sugar-manipulating enzymes including glutamine:fructose-6-phosphate amidotransferase (GFAT), UDP-glucose dehydrogenase (UDP-GlcDH), and hyaluronan synthase (HAS). PBCV-1 genes encoding the putative GFAT and UDP-GlcDH enzymes were expressed in Escherichia coli, and both recombinant proteins have the predicted enzyme activity in cell free extracts. These same two genes are transcribed early in PBCV-1 infection, and both genes are widespread among the Chlorella viruses. The products of the reactions catalyzed by these two enzymes are precursors in the biosynthesis of hyaluronan polysaccharide. Previous experiments established that, like the GFAT and UDP-GlcDH genes, the HAS gene is transcribed early and encodes a functional enzyme (DeAngelis, P. L., Jing. W., Graves, M. V., Burbank, D. E., and Van Etten, J. L. (1997) Science 278, 1800-1803). Interestingly, the predicted amino-acid sequences of the PBCV-1 GFAT and UDP-GlcDH enzymes are more similar to bacterial GFAT and UDP-GlcDH enzymes than to their eukaryotic counterparts. In contrast, the amino-acid sequence of the PBCV-1 HAS enzyme more closely resembles eukaryotic enzymes.

Animals↗

Hyaluronan synthase of chlorella virus PBCV-1.

Sequence analysis of the 330-kilobase genome of the virus PBCV-1 that infects a chlorella-like green algae revealed an open reading frame, A98R, with similarity to several hyaluronan synthases. Hyaluronan is an essential polysaccharide found in higher animals as well as in a few pathogenic bacteria. Expression of the A98R gene product in Escherichia coli indicated that the recombinant protein is an authentic hyaluronan synthase. A98R is expressed early in PBCV-1 infection and hyaluronan is produced in infected algae. These results demonstrate that a virus can encode an enzyme capable of synthesizing a carbohydrate polymer and that hyaluronan exists outside of animals and their pathogens.

Amino Acid Sequence↗

Chlorella virus SC-1A encodes at least five functional and one nonfunctional DNA methyltransferases.

Chlorella virus SC-1A encodes at least six DNA methyltransferases (MTases): four N6-methyldeoxyadenine (m6A) MTases, M x CviSI (TGCmA), M x CviSII (CmATG), M x CviSIII (TCGmA) and M x CviSIV (GmATC), one 5-methyldeoxycytosine (m5C) MTase, M x CviSV (approximately RCmCG), and one nonfunctional m5C MTase, M x CviSVI, which is homologous to the MTase M x CviJI [RGmC(T/C/G)] produced by another chlorella virus IL-3A. Genes encoding three of the SC-1A m6A MTases (M x CviSI, M x CviSII, and M x CviSIII) and the nonfunctional m5C MTase were cloned and sequenced. Neither M x CviSI nor M x CviSIII genes hybridized to genes for their respective isomethylomers, M x CviRI and M x CviBIII, from other chlorella viruses. However, the M x CviSII gene hybridized strongly to its M x CviAII isomethylomer gene from virus PBCV-1. Like the prototype chlorella virus PBCV-1, the SC-1A genome contains inverted terminal repeats, one of which is adjacent to the nonfunctional m5C MTase. The three cloned m6A MTase genes are distributed throughout the approx. 345 kb SC-1A genome.

Amino Acid Sequence↗

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.

Amino Acid Sequence↗

Large deletions in antigenic variants of the chlorella virus PBCV-1.

Four spontaneously derived, antigenic variants of chlorella virus PBCV-1 contained 27- to 37-kb deletions in the left end of the 330-kb genome. Two of the mutants, which were serologically identical, had deletions that began from map position 4.9 or 16 and ended at position 42.2 kb. In total, the two deleted regions encoded 28 putative functional open reading frames (ORFs); these deletions probably arose from homologous recombination. The other two mutants, which were serologically identical but distinct from the first two mutants, lacked the entire left terminal 37 kb of the PBCV-1 genome, including an identical 2.2-kb inverted terminal repeat region present at both ends of the wild-type genome. The deleted left end region was replaced by the transposition of an inverted 7.7- or 18.5-kb copy of the right end of the PBCV-1 genome. The region deleted in these two viruses encoded 26 single-copy ORFs, of which 23 were common to those deleted in the first two mutant viruses. The junctions of the deletions/transpositions probably arose from nonhomologous recombination. Taken together, the results indicate that 40.1 kb of single-copy DNA encoding 31 ORFs at the left end of the genome are unnecessary for PBCV-1 replication in Chlorella strain NC64A in the laboratory. The results also indicate that the size of the inverted terminal repeat region in this virus can be highly variable and that the PBCV-1 DNA packaging process tolerates large changes in genome size.

Antigenic Variation↗

Analysis of 43 kb of the Chlorella virus PBCV-1 330-kb genome: map positions 45 to 88.

Forty-three kb of DNA, located at the left end (45 to 88 kb) of the 330-kb Chlorella virus PBCV-1 genome, was sequenced and analyzed. Eighty-six open reading frames (ORFs) 65 codons or longer were identified; 47 were classified as major ORFs. These 47 major ORFs are densely packed on both strands of PBCV-1 DNA. Seventeen of these major ORFs resemble genes in the sequence databases, including three putative gene products involved in manipulating sugars (glucosamine synthetase, GDP-D-mannose dehydratase, and N-acetylglucosaminyltransferase), two transcription factors, beta-1,3-glucanase, aspartate transcarbamylase, ubiquitin carboxy terminal hydrolase, RNA guanyl transferase, an exonuclease, and a helicase. This is the first time some of these putative PBCV-1 genes have been found in a virus genome. One of the transcription factor-like genes contains a type IB self-splicing intron. Since a spliceosomal processed intron was reported previously in the PBCV-1 DNA polymerase gene, PBCV-1 is the first virus known to contain two different types of introns.

Amino Acid Sequence↗

Characterization of Chlorella virus PBCV-1 CviAII restriction and modification system.

A second DNA site-specific (restriction) endonuclease (R.CviAII) and its cognate adenine DNA methyltransferase (M.CviAII) were isolated from virus PBCV-1 infected Chlorella strain NC64A cells. R.CviAII, a heteroschizomer of the bacterial restriction endonuclease NlaIII, recognizes the sequence CATG, and does not cleave CmATG sequences. However, unlike NlaIII, which cleaves after the G and does not cleave either CmATG or mCATG sequences, CviAII cleaves between the C and A and is unaffected by mCATG methylation. The M.CviAII and R.CviAII genes were cloned and their DNA sequences were determined. These genes are tandemly arranged head-to-tail such that the TAA termination codon of the M.CviAII methyltransferase gene overlaps the ATG translational start site of R.CviAII endonuclease. R.CviAII is the first chlorella virus site-specific endonuclease gene to be cloned and sequenced.

Amino Acid Sequence↗

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.CviJI gene was sequenced and a single open reading frame of 1101 bp was identified that could code for a polypeptide of 367 amino acids with a predicted molecular weight of 41,864. M.CviJI contained regions of amino acids which were similar to bacterial cytosine methyltransferases. Eighteen other Chlorella viruses, of 36 tested, contained DNA sequences which hybridized to the M.CviJI gene; DNA from some, but not all, of these 18 viruses also contained 5-methylcytosine in (G/A)GC(T/C/G) sequences.

Amino Acid Sequence↗

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.CviJI and the cognate site-specific endonuclease CviJI. The growth and burst sizes of the mutants and parent virus were identical. Also host nuclear and chloroplast DNAs disappeared from infected cells at the same rates. Thus M.CviJI and CviJI activities are not required for IL-3A replication and CviJI activity is not essential for host DNA degradation.

Azacitidine↗

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. In some combinations the ratio of viruses (as infective centres) recovered differed significantly from the input ratio.

Adhesiveness↗

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. Like the NC64A viruses the Pbi viruses are large polyhedron with a diameter of 140 to 150 nm, are chloroform sensitive, have many structural proteins, and have large dsDNA genomes of at least 300 kb. However, the Pbi viruses are serologically distinct from the NC64A viruses. The five Pbi virus genomes contain 5-methylcytosine, which varied from 14.2 to 43.1% of the cytosine, and two of them also contained N6-methyladenine. DNAs from the Pbi viruses hybridized poorly with the two NC64A virus DNAs and they have a higher guanine plus cytosine content (ca. 46%) than the NC64A virus DNAs (ca. 40%).

Base Composition↗

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. The PBCV-1 receptor was unaffected by extraction with organic solvents, detergents, high salts, or treatment with several proteases as well as the polysaccharide degrading enzymes, cellulase and pectinase. In contrast, acid and alkali treatments of walls at high temperatures and treatment with an enzyme preparation from PBCV-1 lysates destroyed the virus receptor. We suspect that the receptor is a carbohydrate.

Carbohydrates↗

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.

Base Composition↗

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.

Adenosine Triphosphate↗