Biomedical subjects

David D Dunigan

Publications and source records attributed to David D Dunigan.

4 recordsLinked to original sources

PubMed2006

Cloning of Nt.CviQII nicking endonuclease and its cognate methyltransferase: M.CviQII methylates AG sequences.

Chlorella virus NY-2A has a large, highly methylated dsDNA genome (45% of the cytosines are 5-methylcytosine and 37% of the adenines are N(6)-methyladenine). Here, we report the cloning, expression, and characterization of the NY-2A-encoded CviQII nicking-modification (N-M) system. The nicking endonuclease, Nt.CviQII, recognizes R downward arrowAG (R=A or G, downward arrow indicating cleavage site) sequences and cleaves the phosphodiester bond 5' to the adenosine. Because of the difficulty in cloning and expressing the wild-type Nt.CviQII, C-terminal truncation mutants were generated and full-length Nt.CviQII was reconstructed by intein-mediated peptide ligation. The truncation mutants and the reconstructed full-length Nt.CviQII have the same recognition and cleavage specificity as the native enzyme. Full-length and truncated Nt.CviQII produced by a cell-free transcription/translation system have similar reaction rates. The methyltransferase, M.CviQII, was also cloned and expressed. It modifies the adenine in AG doublets of DNA in vitro and in vivo in Escherichia coli. To our knowledge, M.CviQII is the first adenine methyltransferase that recognizes a dinucleotide. Therefore, M.CviQII may be a useful reagent for blocking endonuclease cleavage when restriction sites overlap with AG sequences.

Amino Acid Sequence↗

PubMed2006

Phycodnaviruses: a peek at genetic diversity.

The family Phycodnaviridae encompasses a diverse collection of large icosahedral, dsDNA viruses infecting algae. These viruses have genomes ranging from 160 to 560kb. 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 with several other families of large DNA viruses, referred to as the nucleocytoplasmic large DNA viruses (NCLDV). The genomes of members in three genera in the Phycodnaviridae have recently been sequenced and the purpose of this manuscript is to summarize these data. The viruses have diverse genome structures, some with large regions of non-coding sequence and others with regions of single-stranded DNA. Typically, phycodnaviruses have the coding capacity for hundreds of genes. The genome analyses have revealed in excess of 1000 unique genes, with only 14 homologous genes held in common among the three genera of the phycodnavirses sequenced to date. Thus, the gene diversity far exceeds the number of so-called "core" genes. Little is known about the replication of these viruses, but the consequences of these infections of the phytoplankton have global affects, including altered geochemical cycling and weather patterns.

DNA Viruses↗

PubMed2006

Virion-associated restriction endonucleases of chloroviruses.

Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. The prototype of the genus is Paramecium bursaria chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.

Animals↗

PubMed2003

Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae.

Selected antiapoptotic genes were expressed in baker's yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress. When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies. Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes. These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast. Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults. These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death. In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses. Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.

Animals↗