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USGS2021

Species and population specific gene expression in blood transcriptomes of marine turtles

Background Transcriptomic data has demonstrated utility to advance the study of physiological diversity and organisms’ responses to environmental stressors. However, a lack of genomic resources and challenges associated with collecting high-quality RNA can limit its application for many wild populations. Minimally invasive blood sampling combined with de novo transcriptomic approaches has great potential to alleviate these barriers. Here, we advance these goals for marine turtles by generating high quality de novo blood transcriptome assemblies to characterize functional diversity and compare global transcriptional profiles between tissues, species, and foraging aggregations. Results We generated high quality blood transcriptome assemblies for hawksbill ( Eretmochelys imbricata ) , loggerhead ( Caretta caretta ), green ( Chelonia mydas ), and leatherback ( Dermochelys coriacea ) turtles. The functional diversity in assembled blood transcriptomes was comparable to those from more traditionally sampled tissues. A total of 31.3% of orthogroups identified were present in all four species, representing a core set of conserved genes expressed in blood and shared across marine turtle species. We observed strong species-specific expression of these genes, as well as distinct transcriptomic profiles between green turtle foraging aggregations that inhabit areas of greater or lesser anthropogenic disturbance. Conclusions Obtaining global gene expression data through non-lethal, minimally invasive sampling can greatly expand the applications of RNA-sequencing in protected long-lived species such as marine turtles. The distinct differences in gene expression signatures between species and foraging aggregations provide insight into the functional genomics underlying the diversity in this ancient vertebrate lineage. The transcriptomic resources generated here can be used in further studies examining the evolutionary ecology and anthropogenic impacts on marine turtles.

BMC Genomics↗

USGS2020

Investigating the gene expression profiles of rehabilitated Florida manatees (Trichechus manatus latirostris) following red tide exposure

To investigate a Florida manatee ( Trichechus manatus latirostris ) mortality event following a red tide bloom in Southwest Florida, an RNA sequencing experiment was conducted. Gene expression changes in white blood cells were assessed in manatees rescued from a red tide affected area (n = 4) and a control group (n = 7) using RNA sequencing. The genes with the largest fold changes were compared between the two groups to identify molecular pathways related to cellular and disease processes. In total, 591 genes (false discovery rate <0.05) were differentially expressed in the red tide group. Of these, 158 were upregulated and 433 were downregulated. This suggests major changes in white blood cell composition following an exposure to red tide. The most highly upregulated gene, Osteoclast associated 2C immunoglobulin-like receptor (OSCAR), was upregulated 12-fold. This gene is involved in initiating the immune response and maintaining a role in adaptive and innate immunity. The most highly downregulated gene, Piccolo presynaptic cytomatrix protein (PCLO), was downregulated by a factor of 977-fold. This gene is associated with cognitive functioning and neurotransmitter release. Downregulation of this gene in other studies was associated with neuronal loss and neuron synapse dysfunction. Among the cellular pathways that were most affected, immune response, including inflammation, wounds and injuries, cell proliferation, and apoptosis were the most predominant. The pathway with the most differentially expressed genes was the immune response pathway with 98 genes involved, many of them downregulated. Assessing the changes in gene expression associated with red tide exposure enhances our understanding of manatee immune response to the red tide toxins and will aid in the development of red tide biomarkers.

Florida↗

USGS1989

Sensitivity of endemic Snake River cutthroat trout to acidity and elevated aluminum

Acidic episodes in waters of the western USA, do not last as long and are not as intense as those in the eastern USA, but we found that the native western cutthroat trout Oncorhynchus clarki is sensitive to even brief reductions in pH. In laboratory studies, fish were exposed to acidity (pH 4.5–6.5) alone or in the presence of aluminum during the first 7 d of the freshly fertilized egg, eyed embryo, alevin, or swim-up larva stages of development. Following exposure to acidity and aluminum, eggs and fish were held under control water quality conditions to 40 d posthatch to assess effects of the exposure on subsequent development. Reductions in pH from 6.5 to 6.0 in low-calcium water (1.4 mg/L) did not affect survival, but reduced growth offish in the early life stages. The presence of as little as 50 μg A1/L at low pH further decreased growth and reduced survival. The most sensitive indicators of stress were loss of ions (determined from whole-body sampling) and reduced swimming in alevins, reduction in the ratio of RNA:DNA, feeding inhibition, and pathology of gill tissue in swim-up larvae. A pH of 6.0 and 50 μg Al/L reduced whole-body sodium by 72% and potassium by 50% in alevins. Reductions in the RNA: DNA ratio, correlated with lower growth rates, were observed in swim-up larvae exposed to pH 5.5 and 50 μg Al/L. Exposure to 50 μg Al/L at pH 6.0 reduced swimming activity of alevins by 68% and feeding rates of swim-up larvae by 67%. In the presence of 50 μg A1/L, pathological changes in gill tissue were observed in swim-up larvae exposed to pH 6.0 or less. Although acidification is not widespread in the western USA, cutthroat trout have a narrow margin of safety between conditions that currently exist and those at which pH and aluminum reduce survival and growth.

Transactions of the American Fisheries Society↗

USGS2014

Chemical and biological quality of water in Grand Lake St. Marys, Ohio, 2011-12, with emphasis on cyanobacteria

Grand Lake St. Marys (GLSM) is a shallow lake in northwest Ohio, which is about 9 miles long and 3 miles wide with depths averaging less than 8 feet. Cyanobacteria blooms are common in GLSM, and high concentrations of microcystins—toxins produced by cyanobacteria—have been documented therein. During 2011–12, the U.S. Geological Survey collected 11 sets of water samples at 6 locations in the lake. The water samples were analyzed for concentrations of nutrients, chlorophyll, and microcystin and to determine plankton community structure and abundance. Analysis by quantitative polymerase chain reaction (qPCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to identify the relations between microcystin concentrations and Planktothrix and Microcystis genotypes (toxic versus non-toxic). The qPCR analysis targets deoxyribonucleic acid (DNA) genes and quantifies the potential for toxin production, whereas the qRT-PCR analysis targets ribonucleic acid (RNA) transcripts and quantifies the expression of the toxin gene. Water samples were collected six times at one site for analyses of major ions and trace elements. In addition, field measurements were made to determine transparency, temperature, dissolved oxygen, pH, and specific conductance of the water. GLSM is shallow with a long fetch, which contributes to the warm and turbid water conditions. Secchi-disk measurements generally ranged from 0.2 to 0.3 meters, and summer water temperatures in GLSM frequently exceed 25 degrees Celsius (°C), with peak temperatures greater than 30 °C. Dissolved oxygen readings below 0.5 milligrams per liter (mg/L) occurred at the lake bottom, which can lead to the internal recycling of phosphorus in the lake. Phytoplankton analyses indicated that GLSM is dominated by cyanobacteria with Planktothrix , the dominant genera during 2011–12. Nitrate ranged from 0.19 to 3.23 mg/L, although concentrations in most samples were less than 1 mg/L. Total nitrogen concentrations ranged from 1.86 to 5.42 mg/L. Orthophosphate (as P) concentrations ranged from less than 0.004 to 0.067 mg/L, although concentrations of most samples were less than 0.004 mg/L. Total phosphorus (as P) concentrations ranged from 0.12 to 0.43 mg/L. Microcystin concentrations ranged from 7.3 to 83 micrograms per liter. Microcystin concentrations were correlated to cyanobacteria biovolumes, and to concentrations of one ion (sodium) and three trace elements (molybdenum, antimony, and lithium). Concentrations of toxin genes ( mcyE ) determined by qPCR were consistently low for Microcystis and consistently high for Planktothrix throughout both sampling years. Concentrations of cyanobacteria found by qPCR were correlated to microcystin concentrations, cyanobacteria biovolumes, selected nutrient concentrations, and other parameters. Results from qRT-PCR assays showed that toxin gene expression was predominantly from the genus Planktothrix , and concentrations of the RNA transcript varied throughout the two sampling years. A number of conditions that may play a role in the dominance of Planktothrix and the production of microcystin were identified including water temperature; low-light transmission; low concentrations of silica and manganese; and relatively high concentrations of sodium, sulfate, and the trace elements of strontium, vanadium, and boron.

Ohio↗

USGS1991

Development of a biotinylated DNA probe for detection of infectious hematopoietic necrosis virus

A nonrad~oact~ve DNA probe assay was developed to detect and ~dent~fy infect~ous hernatopoiet~c necrosls virus (IHNV) uslng a dot blot format The probe a synthet~c DNA oligonucleot~de labeled enzymatlcally w~th biotln hybnd~zed spec~f~cally w~th nucleocaps~d mRNA extracted from Infected cells early In the vlrus repl~cation cycle A rap~d guan~dln~um th~ocyanate based RNA extraction method uslng RNAzol B and rn~crocentrifuge tubes eff~c~ently pioduced h~gh qual~ty RNA from 3 commonly used f~sh cell llnes, CHSE-214, CHH-1, and EPC The probe reacted with 6 d~verse ~solates of IHNV, but d~d not react \nth 2 related rhabdovlruses of fish viral hemorrhagic septlcemla vlrus and H~rame rhabdovlrus The b~ot~nylated probe was sensltlve detect~ng plcogram levels of target mRNA Detect~on and ~dentif~cat~on of IHNV requ~red 2d when cells wele lnoculated at n~ultiplic~t~es of infect~on (MOI) greater than 2 Flve days were necessary to detect and identify IHNV In cells lnoculated at a MO1 of 0 0002

Diseases of Aquatic Organisms↗

USGS2019

Isolation, characterization and molecular identification of a novel aquareovirus that infects the endangered fountain darter, Etheostoma fonticola

The fountain darter Etheostoma fonticola (FOD) is a federally endangered fish listed under the US Endangered Species Act. Here, we identified and characterized a novel aquareovirus isolated from wild fountain darters inhabiting the San Marcos River. This virus was propagated in Chinook salmon embryo (CHSE)-214, rainbow trout gonad-2 and fathead minnow cells at 15°C. The epithelioma papulosum cyprini cell line was refractory at all temperatures evaluated. High throughput sequencing technologies facilitated the complete genome sequencing of this virus utilizing ribosomal RNA-depleted RNA extracted from infected CHSE-214 cells. Conventional PCR primer sets were developed for the detection and confirmation of this virus to assist diagnostic screening methods. Phylogenetic analysis suggests this virus belongs to the Aquareovirus A genus. This research provides requisite initial data critical to support hatchery and refugia biosecurity measures for this endangered species.

Diseases of Aquatic Organisms↗

USGS2010

Using Gambusia affinis growth and condition to assess estuarine habitat quality: A comparison of indices

Numerous indices have been used to estimate fish growth and condition however, differences in sensitivity and reliability of the methods have hampered efforts to identify appropriate indicators for routine evaluation of habitat quality in the field. We compared common morphometric (length, weight, somatic growth, length-weight condition) and biochemical (RNA:DNA ratio, relative DNA content, energy density) growth indices on the same wild-caught mosquitofish Gambusia affinis to examine their usefulness as indicators of habitat quality. A laboratory experiment was used to quantify growth rates of wild-caught G. affinis under different feeding treatments. Field studies consisted of both a short-term enclosure experiment (10 d) and weekly (7 wk) fish collections to compare growth indices in managed inflow and reference marshes during a winter/spring freshwater pulse event in upper Breton Sound, Louisiana, USA. Marshes flooded by restored freshwater pulses were capable of producing optimum growth (0.001 g DW d-1 DW = dry weight) and energetically valuable habitat (>6000 cal g-1 DW) for trophic transport. Because of differences in timing of response, morphometric and biochemical indices were generally not directly correlated, but there was clear agreement in direction and magnitude of response. The most striking difference in timing was that biochemical indices (RNA:DNA) responded more slowly to treatments than did morphometric growth indices. While gross patterns are comparable between indicators, differences in sensitivity and response time between indicators suggest that choice of indicator needs to be accounted for in interpretation and analysis of effects. ?? Inter-Research 2010, www.int-res.com.

Marine Ecology Progress Series↗

USGS2003

Vertical structure of the phytoplankton community associated with a coastal plume in the Gulf of Mexico

Low salinity plumes of coastal origin are occasionally found far offshore, where they display a distinct color signature detectable by satellites. The impact of such plumes on carbon fixation and phytoplankton community structure in vertical profiles and on basin wide scales is poorly understood. On a research cruise in June 1999, ocean-color satellite-images (Sea-viewing Wide Field-of-view Sensor, SeaWiFS) were used in locating a Mississippi River plume in the eastern Gulf of Mexico . Profiles sampled within and outside of the plume were analyzed using flow cytometry, HPLC pigment analysis and primary production using 14 C incorporation. Additionally, RubisCO large subunit (rbcL) gene expression was measured by hybridization of extracted RNA using 3 full-length RNA gene probes specific for individual phytoplankton clades. We also used a combination of RT-PCR/PCR and TA cloning in order to generate cDNA and DNA rbcL clone libraries from samples taken in the plume . Primary productivity was greatest in the low salinity surface layer of the plume . The plume was also associated with high Synechococcus counts and a strong peak in Form IA rbcL expression. Form IB rbcL (green algal) mRNA was abundant at the subsurface chlorophyll maximum (SCM), whereas Form ID rbcL (chromophytic) expression showed little vertical structure . Phylogenetic analysis of cDNA libraries demonstrated the presence of Form IA rbcL Synechococcus phylotypes in the plume . Below the plume , 2 spatially separated and genetically distinct rbcL clades of Prochlorococcus were observed. This indicated the presence of the high- and low-light adapted clades of Prochlorococcus. A large and very diverse clade of Prymnesiophytes was distributed throughout the water column, whereas a clade of closely related prasinophytes may have dominated at the SCM. These data indicate that the Mississippi river plume may dramatically alter the surface picoplankton composition of the Gulf of Mexico , with Synechococcus displacing Prochlorococcus in the surface waters.

Gulf of Mexico↗

USGS2013

Prevalence of neutralizing antibodies to rabies virus in serum of seven species of insectivorous bats from Colorado and New Mexico, United States

We determined the presence of rabies-virus-neutralizing antibodies (RVNA) in serum of 721 insectivorous bats of seven species captured, sampled, and released in Colorado and New Mexico, United States in 2003-2005. A subsample of 160 bats was tested for rabies-virus RNA in saliva. We sampled little brown bats (Myotis lucifugus) at two maternity roosts in Larimer County, Colorado; big brown bats (Eptesicus fuscus) at three maternity roosts in Morgan County, Colorado; and big brown bats at five maternity roosts in Larimer County. We also sampled hoary bats (Lasiurus cinereus) and silver-haired bats (Lasionycteris noctivagans) captured while drinking or foraging over water in Bernalillo County, New Mexico and at various locations in Larimer County. Big brown bats, little brown bats, long-legged myotis (Myotis volans), long-eared myotis (Myotis evotis), and fringed myotis (Myotis thysanodes) were also sampled over water in Larimer County. All species except long-eared myotis included individuals with RVNA, with prevalences ranging from 7% in adult female silver-haired bats to 32% in adult female hoary bats. None of the bats had detectable rabies-virus RNA in oropharyngeal swabs, including 51 bats of 5 species that had RVNA in serum. Antibody-positive bats were present in nine of the 10 maternity colonies sampled. These data suggest that wild bats are commonly exposed to rabies virus and develop a humoral immune response suggesting some degree of viral replication, but many infections fail to progress to clinical disease.

Colorado;New Mexico↗

USGS2019

Relative abundance and molecular evolution of Lake Sinai Virus (Sinaivirus) clades

Lake Sinai Viruses (Sinaivirus) are commonly detected in honey bees ( Apis mellifera ) but no disease phenotypes or fitness consequences have yet been demonstrated. This viral group is genetically diverse, lacks obvious geographic structure, and multiple lineages can co-infect individual bees. While phylogenetic analyses have been performed, the molecular evolution of LSV has not been studied extensively. Here, I use LSV isolates from GenBank as well as contigs assembled from honey bee Sequence Read Archive (SRA) accessions to better understand the evolutionary history of these viruses. For each ORF, substitution rate variation, codon usage, and tests of positive selection were evaluated. Outlier regions of high or low diversity were sought with sliding window analysis and the role of recombination in creating LSV diversity was explored. Phylogenetic analysis consistently identified two large clusters of sequences that correspond to the current LSV1 and LSV2 nomenclature, however lineages sister to LSV1 were the most frequently detected in honey bee SRA accessions. Different expression levels among ORFs suggested the occurrence of subgenomic transcripts. ORF1 and RNA-dependent RNA polymerase had higher evolutionary rates than the capsid and ORF4. A hypervariable region of the ORF1 protein-coding sequence was identified that had reduced selective constraint, but a site-based model of positive selection was not significantly more likely than a neutral model for any ORF. The only significant recombination signals detected between LSV1 and LSV2 initiated within this hypervariable region, but assumptions of the test (single-frame coding and independence of substitution rate by site) were violated. LSV codon usage differed strikingly from that of honey bees and other common honey-bee viruses, suggesting LSV is not strongly co-evolved with that host. LSV codon usage was significantly correlated with that of Varroa destructor , however, despite the relatively weak codon bias exhibited by the latter. While codon usage between the LSV1 and LSV2 clusters was similar for three ORFs, ORF4 codon usage was uncorrelated between these clades, implying rapid divergence of codon use for this ORF only. Phylogenetic placement and relative abundance of LSV isolates reconstructed from SRA accessions suggest that detection biases may be over-representing LSV1 and LSV2 in public databases relative to their sister lineages.

PeerJ↗

USGS2021

Transcriptome assembly and differential gene expression of the invasive avian malaria parasite Plasmodium relictum in Hawaiʻi

The malaria parasite Plasmodium relictum (lineage GRW4) was introduced less than a century ago to the native avifauna of Hawaiʻi, where it has since caused major declines of endemic bird populations. One of the native bird species that is frequently infected with GRW4 is the Hawaiʻi ʻamakihi ( Chlorodrepanis virens ). To achieve a better understanding of the transcriptional activities of this virulent parasite, we performed a controlled challenge experiment of 15 ʻamakihi that were infected with GRW4. Blood samples containing malaria parasites were collected at two time points (intermediate and peak infection stages) from host individuals that were either experimentally infected by mosquitoes or inoculated with infected blood. We then used RNA sequencing to assemble a high‐quality blood transcriptome of P. relictum GRW4, allowing us to quantify parasite expression levels inside individual birds. We found few significant differences (one to two transcripts) in GRW4 expression levels between host infection stages and between inoculation methods. However, 36 transcripts showed differential expression levels among all host individuals, indicating a potential presence of host‐specific gene regulation across hosts. To reduce the extinction risk of the remaining native bird species in Hawaiʻi, genetic resources of the local Plasmodium lineage are needed to enable further molecular characterization of this parasite. Our newly built Hawaiian GRW4 transcriptome assembly, together with analyses of the parasite's transcriptional activities inside the blood of Hawaiʻi ʻamakihi, can provide us with important knowledge on how to combat this deadly avian disease in the future.

Hawaii↗

USGS2017

Infectious hematopoietic necrosis virus virological and genetic surveillance 2000–2012

Surveillance records of the acute RNA pathogen of Pacific salmonid fish infectious hematopoietic necrosis virus are combined for the first time to enable landscape-level ecological analyses and modeling. The study area is the freshwater ecosystems of the large Columbia River watershed in the U.S. states of Washington, Oregon, and Idaho, as well as coastal rivers in Washington and Oregon. The study period is 2000–2012, and records were contributed by all five resource management agencies that operate conservation hatcheries in the study area. Additional records from wild fish were collected from the National Wild Fish Health Survey, operated by the U.S. Fish and Wildlife Survey. After curation and normalization, the data set consists of 6766 records, representing 1146 sample sites and 15 different fish hosts. The virus was found in an average of 12.4% of records, and of these 66.2% also have viral genetic analysis available. This data set is used to conduct univariate ecological and epidemiological analyses and develop a novel hierarchical landscape transmission model for an aquatic pathogen.

Ecology↗

USGS2012

Gene expression, glutathione status and indicators of hepatic oxidative stress in laughing gull (Larus atricilla) hatchlings exposed to methylmercury

Despite extensive studies of methylmercury (MeHg) toxicity in birds, molecular effects on birds are poorly characterized. To improve our understanding of toxicity pathways and identify novel indicators of avian exposure to Hg, the authors investigated genomic changes, glutathione status, and oxidative status indicators in liver from laughing gull (Larus atricilla) hatchlings that were exposed in ovo to MeHg (0.05–1.6 µg/g). Genes involved in the transsulfuration pathway, iron transport and storage, thyroid-hormone related processes, and cellular respiration were identified by suppression subtractive hybridization as differentially expressed. Quantitative polymerase chain reaction (qPCR) identified statistically significant effects of Hg on cytochrome C oxidase subunits I and II, transferrin, and methionine adenosyltransferase RNA expression. Glutathione-S-transferase activity and protein-bound sulfhydryl levels decreased, whereas glucose-6-phosphate dehydrogenase activity increased dose-dependently. Total sulfhydryl concentrations were significantly lower at 0.4 µg/g Hg than in controls. T ogether, these endpoints provided some evidence of compensatory effects, but little indication of oxidative damage at the tested doses, and suggest that sequestration of Hg through various pathways may be important for minimizing toxicity in laughing gulls. This is the first study to describe the genomic response of an avian species to Hg. Laughing gulls are among the less sensitive avian species with regard to Hg toxicity, and their ability to prevent hepatic oxidative stress may be important for surviving levels of MeHg exposures at which other species succumb.

Environmental Toxicology and Chemistry↗

USGS2015

Integrated assessment of wastewater treatment plant effluent estrogenicity in the Upper Murray River, Australia, using the native Murray rainbowfish ( Melanotaenia fluviatilis )

The contamination of major continental river systems by endocrine-active chemicals (EACs) derived from the discharge of wastewater treatment plant (WWTP) effluents can affect human and ecosystem health. As part of a long-term effort to develop a native fish model organism for assessment of endocrine disruption in Australia's largest watershed, the Murray-Darling River Basin, the present study evaluated endocrine disruption in adult males of the native Australian Murray rainbowfish ( Melanotaenia fluviatilis ) exposed to effluent from an activated sludge WWTP and water from the Murray River during a 28-d, continuous-flow, on-site experiment. Analysis of the WWTP effluent and river water detected estrone and 17β-estradiol at concentrations up to approximately 25 ng L −1 . Anti-estrogenicity of effluent samples was detected in vitro using yeast-based bioassays (yeast estrogen screen) throughout the experiment, but estrogenicity was limited to the first week of the experiment. Histological evaluation of the testes indicated significant suppression of spermatogenesis by WWTP effluent after 28 d of exposure. Plasma vitellogenin concentrations and expression of vitellogenin messenger RNA in liver were not significantly affected by exposure to WWTP effluent. The combination of low contaminant concentrations in the WWTP effluent, limited endocrine disrupting effects in the Murray rainbowfish, and high in-stream dilution factors (>99%) suggest minimal endocrine disruption impacts on native Australian fish in the Murray River downstream from the WWTP outfall.

Upper Murray River↗

USGS1993

Sensitivity of early-life stage golden trout to low pH and elevated aluminum

Early-life-stage golden trout ( Oncorhynchus aguabonita aguabonita ) were exposed to acid and Al to examine the response and determine the sensitivity of a western, alpine salmonid to conditions simulating an episodic pH depression. Freshly fertilized eggs, alevins, and swim-up larvae were exposed for 7 d to one of 12 combinations of pH and Al, and surviving fish were held to 40 d post-hatch to determine the effect of exposure on subsequent survival and recovery. Golden trout are sensitive to conditions simulating episodic acidification events typically observed in the field. Significant mortality occurred when the pH of test waters was below 5.0 in the absence of Al or when pH was 5.5 in the presence of 100 μg/L total Al. Behavioral impairments were sensitive indicators of low pH and Al stress. Impaired locomotory and feeding behavior occurred at pH 5.5 without Al and at Al concentrations > 50 μg/L. In contrast, growth, RNA-to-DNA ratio, and whole-body ion concentration were relatively less sensitive indicators of sublethal acid and Al stress.

Environmental Toxicology and Chemistry↗

USGS1999

Cytochrome P4501A induction, benzo[a]pyrene metabolism, and nucleotide adduct formation in fish hepatoma cells: Effect of preexposure to 3,3',4,4',5-pentachlorobiphenyl

In PLHC-1 hepatoma cells, benzo[a]pyrene (B[a]P) caused a maximum induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylation (EROD), after 4 to 8 h of exposure, depending on the B[a]P concentration. The decline of EROD activity at longer exposure times was probably caused by the rapid metabolism of B[a]P in this system (57% metabolism within 4 h incubation). In subsequent experiments, PLHC-1 cells were preinduced with PCB 126 for 24 h and then received a dose of 10, 100, or 1,000 nM 3H-B[a]P. A 1-nM concentration of PCB 126 caused an 80-fold induction of CYP1A activity, resulting in an increase in B[a]P metabolism of less than 10%, except at the highest concentration of B[a]P (1,000 nM), where a 50% increase was observed. In another experiment, an 80-fold induction of CYP1A activity caused a 20% increase in the metabolism of B[a]P (100 nM), and RNA adduct formation was increased approximately twofold. These results indicate that, at exposure concentrations up to 100 nM B[a]P, CYP1A activity is not rate limiting for B[a]P metabolism. Furthermore, CYP1A seems to also he specifically involved in B[a]P activation in PLHC-1 cells. However, CYP1A induction causes only a relatively small increase in activation, probably because of the action of other enzymes involved in B[a]P activation and deactivation.

Environmental Toxicology and Chemistry↗

USGS1993

Polyphosphate present in DNA preparations from fungal species of Collectotrichum inhibits restriction endonucleases and other enzymes

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, 0. C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25°C and to temperatures as high as 100°C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and Taq I DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and Taq I polymerase amplification.

Analytical Biochemistry↗

USGS1999

Sequence features and phylogenetic analysis of the stress protein Hsp90α in chinook salmon Oncorhynchus tshawytscha, a poikilothermic vertebrate

We cloned and sequenced a chinook salmon Hsp90 cDNA; sequence analysis shows it to be Hsp90??. Phylogenetic analysis supports the hypothesis that ?? and ?? paralogs of Hsp90 arose as a result of a gene duplication event and that they diverged early in the evolution of vertebrates, before tetrapods separated from the teleost lineage. Among several differences distinguishing poikilothermic Hsp90?? sequences from their bird and mammal orthologs, the teleost versions specifically lack a characteristic QTQDQP phosphorylation site near the N-terminus. We used the cDNA to develop an RNA (Northern) blot to quantify cellular Hsp90 mRNA levels. Chinook salmon embryonic (CHSE-214) cells responded to heat shock with a rapid rise in Hsp90 mRNA through 4 h, followed by a gradual decline over the next 20 h. Hsp90 mRNA level may be useful as a stress indicator, especially in a laboratory setting or in response to acute heat stress.

Biochemical and Biophysical Research Communication↗