Genomic Organization of Infectious Salmon Anemia Virus

Infectious salmon anemia virus (ISAV) is an emerging pathogen of farmed Atlantic salmon (Salmo salar). The development of an effective ISA virus vaccine is a high priority for salmon producers in the U.S. and elsewhere. The process of developing a recombinant vaccine requires complete genetic characterization of the virus. Toward this end we have cloned, sequenced and determined the organization of the eight segments of single-stranded RNA from ISA virus isolate CCBB. The virus was grown in cell culture and purified by density gradient ultracentrifugation. Viral RNA was isolated from purified ISAV and used in the construction of two different cDNA libraries. After screening the libraries, individual ISA virus-specific cDNA clones were placed into eight groups and the DNA from a representative clone from each group was sequenced. Using Northern blot hybridization results, the eight representative clones were assigned to specific RNA segments of the ISA virus genome and a genetic map of ISA virus strain CCBB was constructed. In addition, N-terminal amino acid sequence analyses of purified ISA virus proteins correlated protein product(s) to specific RNA segments and provided evidence for protein synthesis initiation sites. Finally, Western blot analysis identified viral proteins that were irnmunoreactive with ISA virus-specific serum from mice and Atlantic salmon

Global Gene Expression of Prochlorococcus ecotypes in Response to Changes in Nitrogen Availability

Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium-replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways

S-102 Transfer Pump Restriction Modeling Results

It was determined that a radioactive waste leak in the Hanford S Farm in the vicinity of the S-102 retrieval pump discharge occurred because of over-pressurization and failure of the S-102 dilution water supply hose while operating the retrieval pump in reverse with an obstructed suction cavity and an unobstructed flow path to the dilution water supply hose. This report describes efforts to identify plausible scenarios for the waste leak to occur

Global gene expression of Prochlorococcus ecotypes in response to changes in nitrogen availability

Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (F(v)/F(m)) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium-replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways

Genome-Wide Analysis of Light Sensing in Prochlorococcus

Prochlorococcus MED4 has, with a total of only 1,716 annotated protein-coding genes, the most compact genome of a free-living photoautotroph. Although light quality and quantity play an important role in regulating the growth rate of this organism in its natural habitat, the majority of known light-sensing proteins are absent from its genome. To explore the potential for light sensing in this phototroph, we measured its global gene expression pattern in response to different light qualities and quantities by using high-density Affymetrix microarrays. Though seven different conditions were tested, only blue light elicited a strong response. In addition, hierarchical clustering revealed that the responses to high white light and blue light were very similar and different from that of the lower-intensity white light, suggesting that the actual sensing of high light is mediated via a blue-light receptor. Bacterial cryptochromes seem to be good candidates for the blue-light sensors. The existence of a signaling pathway for the redox state of the photosynthetic electron transport chain was suggested by the presence of genes that responded similarly to red and blue light as well as genes that responded to the addition of DCMU [3-(3,4-dichlorophenyl)-1,1-N-N′-dimethylurea], a specific inhibitor of photosystem II-mediated electron transport

Short RNA half-lives in the slow-growing marine cyanobacterium Prochlorococcus

Background: RNA turnover plays an important role in the gene regulation of microorganisms and influences their speed of acclimation to environmental changes. We investigated whole-genome RNA stability of Prochlorococcus, a relatively slow-growing marine cyanobacterium doubling approximately once a day, which is extremely abundant in the oceans. Results: Using a combination of microarrays, quantitative RT-PCR and a new fitting method for determining RNA decay rates, we found a median half-life of 2.4 minutes and a median decay rate of 2.6 minutes for expressed genes - twofold faster than that reported for any organism. The shortest transcript half-life (33 seconds) was for a gene of unknown function, while some of the longest (approximately 18 minutes) were for genes with high transcript levels. Genes organized in operons displayed intriguing mRNA decay patterns, such as increased stability, and delayed onset of decay with greater distance from the transcriptional start site. The same phenomenon was observed on a single probe resolution for genes greater than 2 kb. Conclusions: We hypothesize that the fast turnover relative to the slow generation time in Prochlorococcus may enable a swift response to environmental changes through rapid recycling of nucleotides, which could be advantageous in nutrient poor oceans. Our growing understanding of RNA half-lives will help us interpret the growing bank of metatranscriptomic studies of wild populations of Prochlorococcus. The surprisingly complex decay patterns of large transcripts reported here, and the method developed to describe them, will open new avenues for the investigation and understanding of RNA decay for all organisms.Deutsche Forschungsgemeinschaft (SPP 1258)German-Israeli Foundation for Scientific Research and Development (young investigator grant 2167-1743.9/2007)Geosciences Research Program (United States. Dept. of Energy)National Science Foundation (U.S.)Gordon and Betty Moore FoundationIsraeli Science Foundation (Morasha grant 1504/06)Fundação para a Ciência e a Tecnologia (IBB/CBME, LA, FEDER/POCI 2010