Validation of ( sup 3 H)thymidine incorporation and its application to detecting natural transformation in the marine environment
Thesis/Dissertation
·
OSTI ID:6828014
The ({sup 3}H)thymidine incorporation method to estimate bacterial DNA synthesis and heterotrophic production was examined by investigating the four major factors and assumptions associated with the technique. When compared to fluorometrically determined rates of DNA synthesis, ({sup 3}H)thymidine incorporation consistently underestimated DNA synthesis by 6 to 8-fold, indicating the inability of ({sup 3}H)thymidine incorporation and isotope dilution assays to accurately determine the amount of thymine bases incorporated into DNA. Non-specific labeling of macromolecules other than DNA was ubiquitous and the percentage of radioactivity incorporated into DNA was inversely related to total rates thymidine incorporation but independent of any other parameter examined. The use of specific inhibitors and a comparison of (methyl-{sup 3}H)thymidine with (6-{sup 3}H)thymidine indicated that non-specific labelling was not the result of a demethylation reaction but the result of ({sup 3}H)thymine catabolism. Four of the 41 marine bacterial isolates examined were incapable of incorporating thymidine into DNA and lacked thymidine transport and thymidine kinase activity. Transformation of an E. coli tdk into one of these organisms resulted in high levels of thymidine kinase activity but no capacity to incorporate or transport thymidine by this organism.
- Research Organization:
- University of South Florida, Tampa, FL (USA)
- OSTI ID:
- 6828014
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
14 SOLAR ENERGY
140505 -- Solar Energy Conversion-- Photochemical
Photobiological
& Thermochemical Conversion-- (1980-)
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AZINES
BACTERIA
CHROMATOGRAPHY
DNA REPLICATION
ENZYME ACTIVITY
ENZYMES
GAS CHROMATOGRAPHY
GENETIC ENGINEERING
GROWTH
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
METABOLISM
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PYRIMIDINES
RIBOSIDES
SEPARATION PROCESSES
THYMIDINE
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
140505 -- Solar Energy Conversion-- Photochemical
Photobiological
& Thermochemical Conversion-- (1980-)
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AZINES
BACTERIA
CHROMATOGRAPHY
DNA REPLICATION
ENZYME ACTIVITY
ENZYMES
GAS CHROMATOGRAPHY
GENETIC ENGINEERING
GROWTH
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
METABOLISM
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PYRIMIDINES
RIBOSIDES
SEPARATION PROCESSES
THYMIDINE
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS