Effect of 5-Fluoro-2′-Deoxyuridine on [
            <sup>3</sup>
            H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [ 3 H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [ 3 H]thymidine incorporation and in [ 3 H]thymidine incorporation per cell. The concentrations that inhibited [ 3 H]thymidine incorporation by 50% ranged from 3 to 11 mg liter −1 for 3,5-dichlorophenol, 6 to 10 mg liter −1 for 2,4-dinitrophenol, and 21 to 123 mg liter −1 for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [ 3 H]leucine incorporation into bacterial protein were similar or larger than those obtained from [ 3 H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [ 3 H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [ 3 H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.

Section: Discussionmentioning

confidence: 99%

Effects of Toxic Substances on Natural Bacterial Assemblages Determined by Means of [ ³ H]Thymidine Incorporation

Riemann¹,

Lindgaard-Jørgensen²

1990

“…The accuracy of the technique, however, is contingent on four factors and assumptions: (i) the need to assess and correct for isotope dilution; (ii) the need to determine the amount of radioactive label incorporated specifically into DNA; (iii) the assumption that all bacteria in the sample are capable of incorporating thymidine; and (iv) the empirical relationship between thymidine incorporation and DNA synthesis (13). Previous studies have examined isotope dilution (2,3,12,17,18,25) and nonspecific macromolecular labeling (9,12,29,31; K. Carmen, F. C. Dobbs, and J. B. Guckert, Limnol.…”

mentioning

confidence: 99%

“…DNA concentration is determined against known standards. For calibration of thymidine incorporation with DNA synthesis in natural waters, samples were taken from Bayboro Harbor, a eutrophic embayment of Tampa Bay in St. Petersburg, Fla. (11,12,22), and oligotrophic offshore waters of the southeast Gulf of Mexico (26°40.81' N, 83058.33' W).…”

mentioning

confidence: 99%

“…The apparent explanation for these results is that isotope dilution analysis (2,3,12,17,18 (6,30). Several studies have examined its use in environmental samples (2,3,12,17,18,25), and the method appears to function much as it does when applied to mammalian tissue cells. These previous studies have demonstrated that isotope dilution analysis accounts for some intraand extracellular dilution of the isotope, although the amount for environmental samples was rarely more than the equivalent of a few nanomolar thymidine.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Underestimation of DNA Synthesis by [ ³ H]Thymidine Incorporation in Marine Bacteria

Jeffrey

Paul

1988

Self Cite

A direct comparison of [3H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [3HJthymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [3HJthymidine incorporation consistently underestimated DNA synthesized by sixto eightfold. These results indicate that although the relationship of [3HJthymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [3H]thymidine incorporation and isotope dilution assays.

“…[3H]thymidine incorporation is the most widely used means by which bacterial activity and productivity are estimated in water column (13,16,24) and sediment samples (4,12,39). Previous studies have examined the effects on thymidine incorporation of isotope dilution (2,18,26,35) and nonspecific labeling of macromolecules other than DNA (4,16,18,37), as well as the relationship between [3H]thymidine incorporation and DNA synthesis (19). One of the essential assumptions for the validity of the [3H]thymidine incorporation method used to estimate bacterial heterotrophic production is that all of the bacteria in the sample are capable of incorporating thymidine into DNA.…”

mentioning

confidence: 99%

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

Jeffrey

Paul

1990

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.