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The incorporation of tritiated thymidine into dna as a measure of the activity of soil micro-organisms
THE INCORPORATION OF TRITIATED THYMIDTNE INTO DNA AS A MEASURE OF THE ACTIVITY OF SOIL MICRO-ORGANISMS
D. R. THOMAS,J. A. RINARDSON and R. J. DICKER Department of Plant Biology. University of Newcastle upon Tyne. NEI 7RC England
Summar?_A
known weight of fresh soil was incubated with 3H-Tdr of known activity and some of the labelled thymidine was incorporated into the microbial DNA. Then the DNA was extracted from the sample and its radioactivity counted in solution. The incorporation of “H-Tdr may be used as a measure of the activity of soil microbes
INTRODl’CTIOU
When spoilt land is being reclaimed for amenity purposes a principal aim is to quickly establish a maintenance-free grass sward on the regraded land. In the absence of toxic substances this can be achieved by sowing a suitable mixture of seeds, fertilizer and wood pulp directly onto the surface (Richardson, Shenton and Dicker. 1971; Richardson and Dicker. 1972). At the end of the first season, and thereafter annually, the shoots and roots of the sward-forming species supply large quantities of organic residues which are essential for soil formation and which are the primary source of food for soil micro-organisms. At a site in County Durham observations were made of the changes taking place in soil properties during sward formation and amongst these were the changes occurring in the soil micro-population. The method described below is simple. rapid and free from some of the inadequacies of existing methods used to monitor the soil micro-population. In an actively dividing population of soil microorganisms there is a synthesis of protoplasm which is complementary to cell division. Synthesis of DNA. which precedes cell division, requires a supply of deoxynucleosides. Thymidine is the deoxynucleoside which is utilized in DNA synthesis but not normally in that of RNA. Consequently the incorporation of thymidine into DNA may be used to indicate the activity of the microbial population. Brock (1967) used thymidine autoradiography to assess bacterial growth rate in the sea and discussed the appliiation of this technique in the analysis of microbial growth rates (Brock. 1971). In the method described here a known weight of soil was incubated with radioactive thymidine of known activity. and then the amount of labelled thymidine that was incorporated into microbial DNA was determined. Thus it was possible to obtain a measure of the
growth activity of at least part of the micro-organisms in the soil. MATERIALS AND
METHODS
(a) In preliminary experiments with garden loam the soil was loosely packed into glass tubing. 15 mm id. x 40 mm. The soil was saturated with sterile distilled water and allowed to drain to field capacity. Then 0.2 ml sterile 3H-Tdr solution (8% x IOH cpm,/ml) was added to the surface of the soil and the soils incubated for various intervals at room temperature. In these experiments the DNA was chemically precipitated in the soil. The precipitated DNA and soil were collected by centrifugation and washed successively to remove excess “H-Tdr. The DNA was counted in the presence of the soil. Results obtained with this method were totally unreliable as the high specific activity thymidine remained in contact with only a small volume of soil even when the tubes were shaken vigorously. There were also indications in the results that much of the isotope remained as unchanged ‘H-Tdr adsorbed on the soil particles. thus the use of total counts remaining in the soil was rendered invalid as an indicator of DNA synthesis. It is possible that if 3H-Tdr was included in the distilled water required to bring the soil to field capacity more reliable results would be obtained. The cost of 3H-Tdr would preclude this procedure for routine analyses. (b) In the next method the 3H-Tdr was diluted with sterile distilled water and added to garden soil contained in a weighing bottle. To provide control samples, soil was sterilized by autoclaving at 120°C for 15 min and known amounts of this sterile soil transferred to the weighing bottles. Known volumes of 3H-Tdr
into sterile
solution
and
non-sterile
were added to the soil
“H-Tdr. The method of pr~p~~r~ttioll of DNA from soils must not. therefore. reyuire the use of organic solvents as they might alter the properties of the Millipore filters. An aqueous method. in which cupric nucleates \Qere precipitated, was adopted (Deluca (21crl.. 1953).
sorl
samples
(Table
1) so
that each soil sumple was saturated.
After incubation of (a) was used to provide
the experimental procedure soil samples for counting. The results demonstrated that high activity was rccovered in the sterile soils (Table I). This activity could not be attributed to incorporation of ‘H-Tdr into DNA of living soil micro-organisms hut rather to adsorption of “I_[-Tdr on to soil particles. Also in our preliminary experiments in counting isotopes in the prcsencc of soil a variety of factors affected the count rate. Organic substances present in the soil acted as quenching agents, as did the soil particles themselves. To climinatc these errors and those due to adsorption it was considered essential to extract the DNA from the soil micro-organisms and to count it in solution with no soil residue present. (c) In the next experiments the micro-organisms in the soil were killed after incubation and the soil and the micro-organisms dialysed against water for cxtcndcd periods. The DNA in the sample was then rolubilired (S~hneidcr, 1945) and the supcrn~~~~~ntscollected. The results from all these experiments strongly indicated that unincorporated, free “H-Tdr was not being totally removed from the soil and microorganisms during dialysis, but that substantial quantitics remained in the final supernatant giving high count rates. Further attempts were made to separate the residual “H-Tdr from DNA. The DNA was precipitated in the linal supernatant and various filtration and column separation techniques were employed. Of these Millipore filters proved the most successful. DNA extracted from a mixed microbial culture was passed through Milliporc filter discs. The discs were washed in water, followed by Yj<,(w,Iv) TCA. The discs were stained purplewith Frulgen’sreagcnt, but tiltrateswerc not positive in tests for DNA, showing that DNA was retained on the Millipore filters. Then 1 ml 3H-Tdr was pipetted on to the surface of Millipore filter which was followed with 100 ml S’!:, (w!v) TCA. Slight suction (4 mlgnin) was applied to obtzain a fast filtration rate. The Millipore discs retained a trace of “H-Tdr but when they were washed with 100 ml 2OY,,(w/v) TCA, the “I-I-Tdr VGISremoved. Thus, Millipore filters were established as the most convenient filters for separating DNA from
Soil samples wt’rc collected in the field. placed in scaled containers and returned at once to the laboratory before an cxprrimcnt. Portions of root and other macroscopic living material were picked out by hand and the soil CI‘ISsicvcd through a 2 mm mesh. About 5 g soil was placed in a Pyrex weighing bottle. 35 mm i.d. x 30 mm. and 2 ml ‘H-Tdr solution (99G’O x IO cpm) was added to saturate the soil. The soil was incubated for 3 5 h in the prcscncc of ,‘HTdr at room temperature. Incubation completed. about 5Oml 5”,, (w/v) TCA was used to wash the soil into a I50 ml conical flask. The 5”,, (w,*v) TCA and soil were heated to 90 C and maintained at this temperature for 20 min to hrcitk open the microor~~ll~isrns and dissolve the DNA (Schneider. 1945). After cooling the mixture was centrifuged at X000 g for IO min. The supcrnatant wx retained. The soil pcltct was resuspended in S”,, (w:v) TCA and centrifuged at 8000 y for IO min. The supernatants were hulked and to the resulting solution was added 1:7 of its volume of saturated aqueous copper sulphate. The solution was allowed to stand overnight at room teniper~itLire when the DNA was precipitated as copper nucleate: (Deluca (‘I t/l.. 1953). The final solution was passed through a Millipore filter (port size O+ & 0.05 /Irn) which retained the DNA. The copper nucleate MRS washed under gentle suction (4 ml~*min) with 20 ml distilled water and 5 successive 20 ml aliyuots 20”,, (w:v) TCA to remove free ‘H-Tdr in the filter. The filter was air dried under suction and transferred to a 56 x 3 mm dia scintillation vial. Then I! ml 2-methox~ethnnt,1 and 3 ml of PPO-POPOP scintillation flL;id were added to the vial. The PPO-POPOP scintillator consisted of ii solution of 5 g. I.%diphenyloxarolc (PPO) and 0.3 g. 1.Gdi-7 (5 phenyloxasolyl)-benrcnc (POPOP) dissolved in I fitrc Analar tolucnc. The methoxyethanol was added to dissolve the Milliporc filters and to absorb any water. The addition of methoxycthanol greatly improved the count rate. prcsumably because it reduced quenching by residual welter and the filter itself. Also it would aid dispersal of ‘HDNA impregnated on the filter. The radioactive samples were counted in a Packard Tri-Garb model 3000 liquid scintillation spectromctcr. Chemicals wcrc purchased from B.D.H. Ltd.. thymidint-(methyl-H,) (“H-Tdr) from the Radiochemical C’cntre. Amcrsham, and PPO and POPOP from Nuclear Enterprises Ltd.. Edinburgh. Millipore titter discs were obtained from the Millipore Corporation. Bedford. Mass. Using this method an experiment was performed with a loam soil from a permanent pasture and a young soil developing on ;I grassed area of ;I reclnimcd
Incorporation
of ‘H-thynidinc
Table 2. Standard
maximum rate of incorporation was in the first 3 hours of incubation. The samples incubated in unsealed bottles, for both loam and shale incorporated more “HTdr into DNA than the samples’ in a limited supply of air. The loam soils incorporated larger amounts of ‘HTdr into DNA than did the shalt. demonstrating that micro-organisms wcrc dividing more rapidly in the loam soil.
deviation in count rates of results presented in Fig. I. Each result 1s the mean of two replicates and counts per min are corrected for background
tion time (h)
Counts, mill,’ g soil and SD
Shale (sealed bottles) S2
0 3 24
30.3 k 2.6 209.0 * 5.0 215.x + 5.0
Shale (unsealed
0 3 23
53.3 + 3.0 176.0 k 5.1 346.5 & 6.7
0 3 21
38.8 +- 2.8 332.1 f 6.1 337.7 * 6.9
0
57.9 * 3.1 725.2 * x.7 812.7 + _ 9.’
Incuba-
Soil
hottles)Sl
Loam (sealed bottles)
Loam (unscaled
L2
bottles) L I
3 24
295
by soil micro-organisllls
Sagan ( lY65) and C’ookc ( 1966) have reportod that Tdr will not be incorporated into the DNA of, organisms which do not possess the cnrymc Tdr-kinasc (ATP:th>midinc 5’ - phosphotransfcrasc. E.C. 2.7. I.21 for the bacterial enl-yme). However. organisms lacking this enzyme arc said to hc few in number (Clzavcr. 1967). When organisms which possess Tdr-kinase arc supplied with Tdr. the Tdr is converted to TTP which is thon assembled, together with other nuclcotides. into DNA. If an organism has Iargc pools of TTP accessible to thymidine-kinasc sites. the thymidine-kinasc may be inhibited (Okazaki and Kornherg. IY64) and low incorporation of Tdr into DNA would result. When ‘H-Tdr is supplied to :I mixed population of micro-organisms in soil it might bc taken up by a proportion of micro-organisms and be incorporated into replicating DNA. Also ‘H-Tdr might he taken up by sonic micro-organisms and he dcgradcd b) Tdr-phosphorylasc to “H-thvminc and dcoxyrihosc-l-phosphate. The “H-thqn&e may hc incorporated into other metabolic mxlducts as \vcll as into DNA. Other micro-organisms might not take up ‘H-Tdr at all. Nevertheless. the method finally adopted provides
colliery site. 5 g samples were incubated for 0. 3 and 24 h in sealed and unsealed bottles. This was to compare the effects of incubation in a limited and a free air supply. The results arc presented in Table 2 and Fig. I. The counts obtained at 0 h. between 30 and 5X cpm,’ g soil, indicate that traces of unchanged 3H-Tdr remained on the filters. However, these quantities of ‘H-Tdr were small and constant and did not affect the overall picture which emerged from the results. It may be concluded that 3H-Tdr was incorporated into the replicating DNA of micro-organisms and that the
L2 SI
s2
I
I
I
3
6
I 9
12
Incubation
I
I
15
16
time.
h
21
t *4
296
D. R. TIIOMAS. J. A. RI(.IIARINI\
relatively easily another parameter of the activity ol micro-organisms in the soil and as such is considered to be of use. That the expected differences in incorporation occurred between loam and shale soils (Table 2) is corroboration of this conclusion. Thus, for example. the method would find application in assessing the growth of at least some part of the soil micro-organism population. It is possible that the enzyme Tdr phosphorylase may exist in free form, active in the soil solution. This enzyme might have been released by rupture or death of soil fauna and flora as well as possibly being released by living organisms into the soil. The degradation of 3H-Tdr by this enzyme may account for the lou rate of incorporation after the initial rapid incorporation (see Fig. 1): the available 3H-Tdr in the soil being reduced. It follows in this case that any’ radioactive thymine formed as a result of the degradation cannot be rapidly incorporated into DNA as othcrwisc high counts would be obtained after 24 h. It is known that when “H-Tdr is mixed with soil much “H-Tdr is adsorbed on to the soil particles (set Table I ) even after extraction with IO”,, (wriv) TCA and 70”,, (v/b) ethanol. Much of the “H-Tdr. a highly charged molecule. ma> remain firmly attached to soil particles and not bc readily available to soil micro-organisms. The initial incorporation into DNA (see Fig. 1) would thus be due to uptake of 3H-Tdr from soil water, this readily available source being exhausted in 5 h. Thus, in applications of this method. initial rates of “H-Tdr incorporation are better measured over, at the most. the first 5 Il.
and R. J. DIG LI II
D. R. THOMAS,J. A. RINARDSON and R. J. DICKER Department of Plant Biology. University of Newcastle upon Tyne. NEI 7RC England
Summar?_A
known weight of fresh soil was incubated with 3H-Tdr of known activity and some of the labelled thymidine was incorporated into the microbial DNA. Then the DNA was extracted from the sample and its radioactivity counted in solution. The incorporation of “H-Tdr may be used as a measure of the activity of soil microbes
INTRODl’CTIOU
When spoilt land is being reclaimed for amenity purposes a principal aim is to quickly establish a maintenance-free grass sward on the regraded land. In the absence of toxic substances this can be achieved by sowing a suitable mixture of seeds, fertilizer and wood pulp directly onto the surface (Richardson, Shenton and Dicker. 1971; Richardson and Dicker. 1972). At the end of the first season, and thereafter annually, the shoots and roots of the sward-forming species supply large quantities of organic residues which are essential for soil formation and which are the primary source of food for soil micro-organisms. At a site in County Durham observations were made of the changes taking place in soil properties during sward formation and amongst these were the changes occurring in the soil micro-population. The method described below is simple. rapid and free from some of the inadequacies of existing methods used to monitor the soil micro-population. In an actively dividing population of soil microorganisms there is a synthesis of protoplasm which is complementary to cell division. Synthesis of DNA. which precedes cell division, requires a supply of deoxynucleosides. Thymidine is the deoxynucleoside which is utilized in DNA synthesis but not normally in that of RNA. Consequently the incorporation of thymidine into DNA may be used to indicate the activity of the microbial population. Brock (1967) used thymidine autoradiography to assess bacterial growth rate in the sea and discussed the appliiation of this technique in the analysis of microbial growth rates (Brock. 1971). In the method described here a known weight of soil was incubated with radioactive thymidine of known activity. and then the amount of labelled thymidine that was incorporated into microbial DNA was determined. Thus it was possible to obtain a measure of the
growth activity of at least part of the micro-organisms in the soil. MATERIALS AND
METHODS
(a) In preliminary experiments with garden loam the soil was loosely packed into glass tubing. 15 mm id. x 40 mm. The soil was saturated with sterile distilled water and allowed to drain to field capacity. Then 0.2 ml sterile 3H-Tdr solution (8% x IOH cpm,/ml) was added to the surface of the soil and the soils incubated for various intervals at room temperature. In these experiments the DNA was chemically precipitated in the soil. The precipitated DNA and soil were collected by centrifugation and washed successively to remove excess “H-Tdr. The DNA was counted in the presence of the soil. Results obtained with this method were totally unreliable as the high specific activity thymidine remained in contact with only a small volume of soil even when the tubes were shaken vigorously. There were also indications in the results that much of the isotope remained as unchanged ‘H-Tdr adsorbed on the soil particles. thus the use of total counts remaining in the soil was rendered invalid as an indicator of DNA synthesis. It is possible that if 3H-Tdr was included in the distilled water required to bring the soil to field capacity more reliable results would be obtained. The cost of 3H-Tdr would preclude this procedure for routine analyses. (b) In the next method the 3H-Tdr was diluted with sterile distilled water and added to garden soil contained in a weighing bottle. To provide control samples, soil was sterilized by autoclaving at 120°C for 15 min and known amounts of this sterile soil transferred to the weighing bottles. Known volumes of 3H-Tdr
into sterile
solution
and
non-sterile
were added to the soil
“H-Tdr. The method of pr~p~~r~ttioll of DNA from soils must not. therefore. reyuire the use of organic solvents as they might alter the properties of the Millipore filters. An aqueous method. in which cupric nucleates \Qere precipitated, was adopted (Deluca (21crl.. 1953).
sorl
samples
(Table
1) so
that each soil sumple was saturated.
After incubation of (a) was used to provide
the experimental procedure soil samples for counting. The results demonstrated that high activity was rccovered in the sterile soils (Table I). This activity could not be attributed to incorporation of ‘H-Tdr into DNA of living soil micro-organisms hut rather to adsorption of “I_[-Tdr on to soil particles. Also in our preliminary experiments in counting isotopes in the prcsencc of soil a variety of factors affected the count rate. Organic substances present in the soil acted as quenching agents, as did the soil particles themselves. To climinatc these errors and those due to adsorption it was considered essential to extract the DNA from the soil micro-organisms and to count it in solution with no soil residue present. (c) In the next experiments the micro-organisms in the soil were killed after incubation and the soil and the micro-organisms dialysed against water for cxtcndcd periods. The DNA in the sample was then rolubilired (S~hneidcr, 1945) and the supcrn~~~~~ntscollected. The results from all these experiments strongly indicated that unincorporated, free “H-Tdr was not being totally removed from the soil and microorganisms during dialysis, but that substantial quantitics remained in the final supernatant giving high count rates. Further attempts were made to separate the residual “H-Tdr from DNA. The DNA was precipitated in the linal supernatant and various filtration and column separation techniques were employed. Of these Millipore filters proved the most successful. DNA extracted from a mixed microbial culture was passed through Milliporc filter discs. The discs were washed in water, followed by Yj<,(w,Iv) TCA. The discs were stained purplewith Frulgen’sreagcnt, but tiltrateswerc not positive in tests for DNA, showing that DNA was retained on the Millipore filters. Then 1 ml 3H-Tdr was pipetted on to the surface of Millipore filter which was followed with 100 ml S’!:, (w!v) TCA. Slight suction (4 mlgnin) was applied to obtzain a fast filtration rate. The Millipore discs retained a trace of “H-Tdr but when they were washed with 100 ml 2OY,,(w/v) TCA, the “I-I-Tdr VGISremoved. Thus, Millipore filters were established as the most convenient filters for separating DNA from
Soil samples wt’rc collected in the field. placed in scaled containers and returned at once to the laboratory before an cxprrimcnt. Portions of root and other macroscopic living material were picked out by hand and the soil CI‘ISsicvcd through a 2 mm mesh. About 5 g soil was placed in a Pyrex weighing bottle. 35 mm i.d. x 30 mm. and 2 ml ‘H-Tdr solution (99G’O x IO cpm) was added to saturate the soil. The soil was incubated for 3 5 h in the prcscncc of ,‘HTdr at room temperature. Incubation completed. about 5Oml 5”,, (w/v) TCA was used to wash the soil into a I50 ml conical flask. The 5”,, (w,*v) TCA and soil were heated to 90 C and maintained at this temperature for 20 min to hrcitk open the microor~~ll~isrns and dissolve the DNA (Schneider. 1945). After cooling the mixture was centrifuged at X000 g for IO min. The supcrnatant wx retained. The soil pcltct was resuspended in S”,, (w:v) TCA and centrifuged at 8000 y for IO min. The supernatants were hulked and to the resulting solution was added 1:7 of its volume of saturated aqueous copper sulphate. The solution was allowed to stand overnight at room teniper~itLire when the DNA was precipitated as copper nucleate: (Deluca (‘I t/l.. 1953). The final solution was passed through a Millipore filter (port size O+ & 0.05 /Irn) which retained the DNA. The copper nucleate MRS washed under gentle suction (4 ml~*min) with 20 ml distilled water and 5 successive 20 ml aliyuots 20”,, (w:v) TCA to remove free ‘H-Tdr in the filter. The filter was air dried under suction and transferred to a 56 x 3 mm dia scintillation vial. Then I! ml 2-methox~ethnnt,1 and 3 ml of PPO-POPOP scintillation flL;id were added to the vial. The PPO-POPOP scintillator consisted of ii solution of 5 g. I.%diphenyloxarolc (PPO) and 0.3 g. 1.Gdi-7 (5 phenyloxasolyl)-benrcnc (POPOP) dissolved in I fitrc Analar tolucnc. The methoxyethanol was added to dissolve the Milliporc filters and to absorb any water. The addition of methoxycthanol greatly improved the count rate. prcsumably because it reduced quenching by residual welter and the filter itself. Also it would aid dispersal of ‘HDNA impregnated on the filter. The radioactive samples were counted in a Packard Tri-Garb model 3000 liquid scintillation spectromctcr. Chemicals wcrc purchased from B.D.H. Ltd.. thymidint-(methyl-H,) (“H-Tdr) from the Radiochemical C’cntre. Amcrsham, and PPO and POPOP from Nuclear Enterprises Ltd.. Edinburgh. Millipore titter discs were obtained from the Millipore Corporation. Bedford. Mass. Using this method an experiment was performed with a loam soil from a permanent pasture and a young soil developing on ;I grassed area of ;I reclnimcd
Incorporation
of ‘H-thynidinc
Table 2. Standard
maximum rate of incorporation was in the first 3 hours of incubation. The samples incubated in unsealed bottles, for both loam and shale incorporated more “HTdr into DNA than the samples’ in a limited supply of air. The loam soils incorporated larger amounts of ‘HTdr into DNA than did the shalt. demonstrating that micro-organisms wcrc dividing more rapidly in the loam soil.
deviation in count rates of results presented in Fig. I. Each result 1s the mean of two replicates and counts per min are corrected for background
tion time (h)
Counts, mill,’ g soil and SD
Shale (sealed bottles) S2
0 3 24
30.3 k 2.6 209.0 * 5.0 215.x + 5.0
Shale (unsealed
0 3 23
53.3 + 3.0 176.0 k 5.1 346.5 & 6.7
0 3 21
38.8 +- 2.8 332.1 f 6.1 337.7 * 6.9
0
57.9 * 3.1 725.2 * x.7 812.7 + _ 9.’
Incuba-
Soil
hottles)Sl
Loam (sealed bottles)
Loam (unscaled
L2
bottles) L I
3 24
295
by soil micro-organisllls
Sagan ( lY65) and C’ookc ( 1966) have reportod that Tdr will not be incorporated into the DNA of, organisms which do not possess the cnrymc Tdr-kinasc (ATP:th>midinc 5’ - phosphotransfcrasc. E.C. 2.7. I.21 for the bacterial enl-yme). However. organisms lacking this enzyme arc said to hc few in number (Clzavcr. 1967). When organisms which possess Tdr-kinase arc supplied with Tdr. the Tdr is converted to TTP which is thon assembled, together with other nuclcotides. into DNA. If an organism has Iargc pools of TTP accessible to thymidine-kinasc sites. the thymidine-kinasc may be inhibited (Okazaki and Kornherg. IY64) and low incorporation of Tdr into DNA would result. When ‘H-Tdr is supplied to :I mixed population of micro-organisms in soil it might bc taken up by a proportion of micro-organisms and be incorporated into replicating DNA. Also ‘H-Tdr might he taken up by sonic micro-organisms and he dcgradcd b) Tdr-phosphorylasc to “H-thvminc and dcoxyrihosc-l-phosphate. The “H-thqn&e may hc incorporated into other metabolic mxlducts as \vcll as into DNA. Other micro-organisms might not take up ‘H-Tdr at all. Nevertheless. the method finally adopted provides
colliery site. 5 g samples were incubated for 0. 3 and 24 h in sealed and unsealed bottles. This was to compare the effects of incubation in a limited and a free air supply. The results arc presented in Table 2 and Fig. I. The counts obtained at 0 h. between 30 and 5X cpm,’ g soil, indicate that traces of unchanged 3H-Tdr remained on the filters. However, these quantities of ‘H-Tdr were small and constant and did not affect the overall picture which emerged from the results. It may be concluded that 3H-Tdr was incorporated into the replicating DNA of micro-organisms and that the
L2 SI
s2
I
I
I
3
6
I 9
12
Incubation
I
I
15
16
time.
h
21
t *4
296
D. R. TIIOMAS. J. A. RI(.IIARINI\
relatively easily another parameter of the activity ol micro-organisms in the soil and as such is considered to be of use. That the expected differences in incorporation occurred between loam and shale soils (Table 2) is corroboration of this conclusion. Thus, for example. the method would find application in assessing the growth of at least some part of the soil micro-organism population. It is possible that the enzyme Tdr phosphorylase may exist in free form, active in the soil solution. This enzyme might have been released by rupture or death of soil fauna and flora as well as possibly being released by living organisms into the soil. The degradation of 3H-Tdr by this enzyme may account for the lou rate of incorporation after the initial rapid incorporation (see Fig. 1): the available 3H-Tdr in the soil being reduced. It follows in this case that any’ radioactive thymine formed as a result of the degradation cannot be rapidly incorporated into DNA as othcrwisc high counts would be obtained after 24 h. It is known that when “H-Tdr is mixed with soil much “H-Tdr is adsorbed on to the soil particles (set Table I ) even after extraction with IO”,, (wriv) TCA and 70”,, (v/b) ethanol. Much of the “H-Tdr. a highly charged molecule. ma> remain firmly attached to soil particles and not bc readily available to soil micro-organisms. The initial incorporation into DNA (see Fig. 1) would thus be due to uptake of 3H-Tdr from soil water, this readily available source being exhausted in 5 h. Thus, in applications of this method. initial rates of “H-Tdr incorporation are better measured over, at the most. the first 5 Il.
and R. J. DIG LI II