Browsing by Subject "Polymerase chain reaction"

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Detection, classification, genetic diversity and molecular evolution of algal viruses based on DNA polymerase genes
(1995) Chen, Feng, 1964-; Suttle, Curtis Arnold, 1955-
A polymerase chain reaction (PCR) technique with three highly degenerate primers (group-specific primers AVS1/AVS2 and nested primer POL) was developed to detect and amplify DNA polymerase genes (DNA pol) from 13 virus isolates which infect three genera of distantly related microalgae, Chlorella strains NC64A and Pbi, Micromonas pusilla and Chrysochromulina spp. Amplified DNA pol fragments were cloned and sequenced, and the genetic relatedness among algal viruses examined. Phylogenetic trees based on DNA pol sequences and hybridization of viral genomic DNA showed similar branching patterns. Genetic relatedness calculated from the hybridization and sequencing data showed a good concordance (r=0.90), indicating that DNA pol can be used to determine genetic relatedness and infer phylogenetic relationships among these viruses, and potentially among other organisms. It was found that nucleotide sequences of DNA pol share higher similarities among algal viruses than with other organisms, and viruses infecting the same species had higher sequence similarities than those infecting different species. Sequence analysis of inferred amino acid sequences of DNA pol from 24 double-stranded DNA viruses indicated that viruses which infect these microalgae form a distinct phyletic group, and are more closely related to herpes viruses than to other dsDNA viruses. The branching patterns of the phylogenetic tree corresponded well with groupings based on the International Committee on Taxonomy of Viruses. The study suggests that these algal viruses share a common ancestor and belong to the Phycodnaviridae. Nested PCR was also used to amplify DNA pol genes from natural virus communities concentrated by ultrafiltration and centrifugation, from the Gulf of Mexico. DNA pol genes from algal viruses were detected in 5 of 6 stations. The PCR products from an offshore station (station B11) were cloned, analyzed by restriction fragment length polymorphism (RFLP) and representative clones sequenced to examine the genetic diversity of algal viruses in nature. The five RFLP patterns or operational taxonomy units (OTUs) that were observed ranged from 9 to 34% of the 33 clones in the library. Based on sequence analysis four OTUs were closely related to the Micromonas viruses, whereas one OTU was an unknown algal virus
Linking gene expression to performance in a fungal vapor-phase bioreactor treating ethylbenzene
(2004) Gunsch, Claudia Kneller; Kinney, Kerry A.
Large quantities of volatile organic compounds (VOCs) are emitted from industrial sources into the atmosphere every year. Fungal biofiltration has been demonstrated to be an attractive treatment option for VOCs. In this technology, contaminated air is passed through a biologically active packed bed where the microorganisms degrade the pollutants to benign products. Most biofilter research conducted to date has relied solely on macroscale monitoring such as degradation profiles and nitrogen availability. While these macroscale parameters are important for system performance, they neglect molecular level biodegradation mechanisms in the biofilm. In this research, quantitative real-time reverse transcription polymerase chain reaction (qRT PCR) was used to quantify gene expression variations in the biofilm. The objective of this research was to assess the utility of the qRT-PCR tool for linking microscale gene expression to macroscale bioreactor performance and optimization. The model system used was a biofilter inoculated with the fungus Exophiala lecanii-corni treating ethylbenzene. A comparative threshold method was employed to quantify the relative gene expression of the target gene to a housekeeping gene (18S rRNA). A portion of a key gene involved in ethylbenzene metabolism (ElHDO – homogentisate dioxygenase) was isolated and its gene expression monitored as a function of substrate feed, nutrient concentration and transient loading conditions. In batch experiments, qRT-PCR effectively described changes in relative gene target expression numbers (TN) as a function of substrate mixtures and nutrient concentrations. In the biofilter, TN was found to be a leading indicator of bioreactor failure when a repressor compound was introduced into the column feed. During the transient feed experiment, ElHDO expression slowly shutdown over a 24-hour time period when the ethylbenzene feed was discontinued, but rapidly recovered upon its re-introduction to the system. Overall, qRT-PCR provides valuable insights into the microscale phenomena occurring in the biofilm. However, this method may not be well suited to describe the effect of operational modifications which cause only small gene expression changes due to its low sensitivity under such conditions.