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The effect of starvation on the nucleotide composition of the ribonucleic acid in Amoeba proteus
418
PRELIMINARY NOTES
In conclusion, the pulse-labelled RNA, if handle~t under conditions where degradation is properly prevented, can be fractionated into several RNA factions having considerably high sedimentation coefficients either by column chromatography on methylated serum albumin or b y sucrose density-gradient centrifugation. All pulselabelled RNA fractions have a base ratio equivalent with that of E. coli DNA. The role and fate of these RNA's are now being studied. Very recently, HAYASHI AND SPIEGELMAN11 reported that the selective synthesis of the RNA similar to that reported in this communication takes place under special culture conditions. The preparation obtained by their method also included RNA's with sedimentation coefficients considerably larger than the 8-S value previously reported. The valuable advice and help of Drs. A. ISHIHAMA, E. OTAKA and H. MITSUI in our laboratory are gratefully acknowledged. This work was aided b y grants from the Rockefeller Foundation, the Asahi Press and the Ministry of Education of Japan.
Institute /or Molecular Biology, and Biological Institute, Faculty o/ Science, Nagoya University, Chikusa-Ku, Nagoya (Japan)
M. TAKAI N. KONDO S. OSAWA
1 F. GROS, H. HIATT, \'V. GILBERT, C. G. I(URLAND, R. \~7. t{ISEBROUGH AND J. D. WATSON, Nature, 19o (1961) 581. J. MCCARTHY, R. J. BR1TTEN AND R. B. ROBERTS, Biophys. J., (1962) in the press. 3 j . D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. 4 L. PHILIPSON, J. Gen. Physiol., 44 (1961) 899. R. J. BRITTEN AND R. B. ROBERTS, Science, 131 (196o) 32. 8 C. G. I(URLA,~D, J. Mol. Biol., 2 (196o) 3" "I S. OSAXVA, ]['~. TAK.~.TA AND Y. HOTTA, Biochim. Biophys. Acla, 28 (1958) 271. S. OSAIVA, I. \VATANABE, A. ISHIHAMA AND H. MITSUI, Biochem. Biophys. Research Communs., (1962) in the press. 9 A. N. BELOZERSKY AND A. S. S. SPIRIN, in E. CHARGAFF AND J. N. DAVlDSON, The Nucleic Acids, Vol. I I I , Academic Press Inc., New York and London, 196o, p. 147. 10 S. OSAWA, Biochim. Biophys. ,4eta, 42 (196o) 244. H M. HAVASHI AND S. SPIEGELMAN, Proc. Natl..4cad. Sci. U.S., 47 (1961) 1564.
Received December I l t h , 1961 Biochim. Biophys. Acta, 55 (1962) 416-418
The effect of storvotion on the nucleotide composition of the ribonucleic acid in A m o e b a profeus
Amoeba proteus has been used extensively as a subject for studies aimed at elucidating the function and behaviour of RNA. These investigations have been limited by the absence of information on the composition of the RNA in this organism and complicated by the fact that under normal culture conditions the amoeba m a y contain appreciable amounts of foreign RNA for unknown periods of time. This RNA is introduced into the amoeba by the ingestion of live protozoa which make up the bulk if not all of the amoeba's diet. Under these circumstances it was thought expedient to establish the composition of tile RNA of the amoeba at different times after the introduction Biochim. Biophys. Acta, 55 (1962) 418-42o
PRELIMINARY NOTES
419
of the food organism. The readily observable ingestion of food as well as the relatively long survival time of A. proteus under starvation conditions made this approach feasible 1. Amoeba proteus cells were maintained in plastic dishes (15o×25 mm, Falcon Plastics) in a dilute salt solution* and fed with washed Tetrahymena pyri[ormis (strain W, obtained from G. W. Kidder, Amherst University, and grown as pure culture on 2 % proteose peptone) as described b y GRIFFINK When the amoeba cultures reached a density of approx. 400 cells/cm* they were fed with an excess of tetrahymena so that all cells were visibly rounded b y about I h after the administration of the food organism. The cells were washed free of uningested t e t r a h y m e n a and prepared for RNA analysis either immediately (freshly fed samples) or after 4 or IO days of starvation. Cell samples for analysis were concentrated in I5-ml centrifuge tubes b y subjecting them to three 5-min centrifugations at IOOO×g. From o.3-1.2 cm 3 of packed amoebae were used for each determination (according to GRIFI~IN3 these volumes should contain between I - l O s and 6. lO s cells). T e t r a h y m e n a samples were prepared for analysis b y centrifuging pure cultures, washing twice in amoeba medium, and then proceeding as for a m o e b a samples. The method of nucleotide analysis used was that of OSAWA et al. 4. lJltravioletabsorption measurements were carried out in a Zeiss spectrophotometer (Model PMQII). Complete spectra were obtained on the pooled peaks after removal of the formic acid in vacuum. The samples ranged in absorbancy at the absorption peak for adenylic acid from o.5oo-1.5oo for amoeba and from 3.00-4.o0 for tetrahymena. The feeding process in A. proteus consists of the ingestion of the living food organism, its enclosure in a food vacuole and its subsequent digestion. If the composition of the RNA of t h e food organism differs significantly from that of the amoeba then the fate of the bulk of this RNA can be followed and the time after ingestion at which its presence is no longer detectable can be established. From this point in starvation on, the observed base composition should represent the composition of the RNA of the amoeba alone. The results of the present analysis, summarized in Table I, indicate t h a t the food TABLE
I
BASE COMPOSITION OF R N A FROM d . proteus AND T. pyri/ormis A. proteus Mole (%)*
T. pyrilormis Freshly ]ed
Ado Gua Cyd Urd
Starved 4 days
Starved
xo days
3o.3~o.3 2o.6~o. 7 22.2io.2 26.7~o. 4
23.7~o. 4 28.2±0.4 26.7ii.I 21.7±1.o
2o.5~I. 5 28.0±0.4 3o.7±1.3 2o.3~o.3
2o.6±1.4 27.2±0.8" 29.5±1.7 22-5~o.8
i.ii
i.oi
I.O 3
i,oo
AT
o.75
1.2o
i .29
1.3o
Purine Pyrimidine
i .0 4
I.o 7
o.9I
0.92
6-Amino 6-Oxo
GC
* T h e s e d a t a are t h e a v e r a g e s of d u p l i c a t e s a m p l e s . Biochim. Biophys. Acta, 55 (i962) 4 1 8 - 4 2 o
420
PRELIMINARY NOTES
organism used, T. pyri/ormis, has an RNA complement whose composition is distinctly different from that of A. proteus. The data are in good agreement with those of SCHERBAUM5 for this strain of tetrahymena. The presence of the foreign RNA is evident in the analysis of A. proteus immediately after feeding, as indicated by the lower GC/AU ratio and b y the high purine/pyrimidine ratio. The difference is absent 4 days later. I t follows that within the limits of precision of our analysis, t e t r a h y m e n a RNA is no longer detectable in the amoeba four days after its introduction. Unpublished experiments performed in our laboratory with t e t r a h y m e n a whose RNA was heavily labeled show that this RNA is not rejected b y the amoeba as such. It is most probable that the RNA of the food organism loses its characteristic identity b y degradation to at least the nucleotide level. (While even cursory examination suggests that amoebae feeding on t e t r a h y m e n a for extended periods do not lose their identity and begin to resemble their prey, it is reassuring to find that this conclusion is not contradicted at the molecular level.) The distribution of RNA-bound Azure B stain in the starved amoebae of the cultures used for chemical analysis was found to be predominantly homogeneous. This observation not only confirms the absence of undigested masses of t e t r a h y m e n a RNA, but further indicates that the possible contribution of any RNA-containing bodies of light-microscope dimension (i.e. yeast or bacteria contained in vacuoles, or symbiotic microorganisms) is negligible in comparison to the mass of RNA from the nucleus and cytoplasm of the amoeba. In summary, the results indicate that the A. proteus RNA is of the G-C type, that of T. pyri/ormis of the A - U type, and that the tetrahymenal RNA is assimilated b y the amoeba before the fourth day after ingestion of the living ciliate. This work was supported by grant No. RG-6317, National Institutes of Health, and by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation.
Department o/ Zoology, University o/ Wisconsin, Madison, Wisc. (U.S.A.) 1 2 3 4 6
J. E. CUMMINS W. PLAUT
A. I. COHEN, J. Biophys. Biochem. Cytol., 3 (1957) 923. D. PRESCOTT AND T. JAMES, Exptl. Cell Research, 8 (1955) 256. j . L. GRIFFIN, Exptl. Cell Research, 21 (196o) 17o. S. OSAWA, K. TAKATA AND Y. HOTTA, Biochim. Biophys. Acta, 28 (1958) 229. O. SCHERBAUM, Exptl. Cell Research, 13 (1957) 24.
Received December I l t h , 1961
Biochim. Biophys. Acta, 55 (1962) 418-42o
PRELIMINARY NOTES
In conclusion, the pulse-labelled RNA, if handle~t under conditions where degradation is properly prevented, can be fractionated into several RNA factions having considerably high sedimentation coefficients either by column chromatography on methylated serum albumin or b y sucrose density-gradient centrifugation. All pulselabelled RNA fractions have a base ratio equivalent with that of E. coli DNA. The role and fate of these RNA's are now being studied. Very recently, HAYASHI AND SPIEGELMAN11 reported that the selective synthesis of the RNA similar to that reported in this communication takes place under special culture conditions. The preparation obtained by their method also included RNA's with sedimentation coefficients considerably larger than the 8-S value previously reported. The valuable advice and help of Drs. A. ISHIHAMA, E. OTAKA and H. MITSUI in our laboratory are gratefully acknowledged. This work was aided b y grants from the Rockefeller Foundation, the Asahi Press and the Ministry of Education of Japan.
Institute /or Molecular Biology, and Biological Institute, Faculty o/ Science, Nagoya University, Chikusa-Ku, Nagoya (Japan)
M. TAKAI N. KONDO S. OSAWA
1 F. GROS, H. HIATT, \'V. GILBERT, C. G. I(URLAND, R. \~7. t{ISEBROUGH AND J. D. WATSON, Nature, 19o (1961) 581. J. MCCARTHY, R. J. BR1TTEN AND R. B. ROBERTS, Biophys. J., (1962) in the press. 3 j . D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. 4 L. PHILIPSON, J. Gen. Physiol., 44 (1961) 899. R. J. BRITTEN AND R. B. ROBERTS, Science, 131 (196o) 32. 8 C. G. I(URLA,~D, J. Mol. Biol., 2 (196o) 3" "I S. OSAXVA, ]['~. TAK.~.TA AND Y. HOTTA, Biochim. Biophys. Acla, 28 (1958) 271. S. OSAIVA, I. \VATANABE, A. ISHIHAMA AND H. MITSUI, Biochem. Biophys. Research Communs., (1962) in the press. 9 A. N. BELOZERSKY AND A. S. S. SPIRIN, in E. CHARGAFF AND J. N. DAVlDSON, The Nucleic Acids, Vol. I I I , Academic Press Inc., New York and London, 196o, p. 147. 10 S. OSAWA, Biochim. Biophys. ,4eta, 42 (196o) 244. H M. HAVASHI AND S. SPIEGELMAN, Proc. Natl..4cad. Sci. U.S., 47 (1961) 1564.
Received December I l t h , 1961 Biochim. Biophys. Acta, 55 (1962) 416-418
The effect of storvotion on the nucleotide composition of the ribonucleic acid in A m o e b a profeus
Amoeba proteus has been used extensively as a subject for studies aimed at elucidating the function and behaviour of RNA. These investigations have been limited by the absence of information on the composition of the RNA in this organism and complicated by the fact that under normal culture conditions the amoeba m a y contain appreciable amounts of foreign RNA for unknown periods of time. This RNA is introduced into the amoeba by the ingestion of live protozoa which make up the bulk if not all of the amoeba's diet. Under these circumstances it was thought expedient to establish the composition of tile RNA of the amoeba at different times after the introduction Biochim. Biophys. Acta, 55 (1962) 418-42o
PRELIMINARY NOTES
419
of the food organism. The readily observable ingestion of food as well as the relatively long survival time of A. proteus under starvation conditions made this approach feasible 1. Amoeba proteus cells were maintained in plastic dishes (15o×25 mm, Falcon Plastics) in a dilute salt solution* and fed with washed Tetrahymena pyri[ormis (strain W, obtained from G. W. Kidder, Amherst University, and grown as pure culture on 2 % proteose peptone) as described b y GRIFFINK When the amoeba cultures reached a density of approx. 400 cells/cm* they were fed with an excess of tetrahymena so that all cells were visibly rounded b y about I h after the administration of the food organism. The cells were washed free of uningested t e t r a h y m e n a and prepared for RNA analysis either immediately (freshly fed samples) or after 4 or IO days of starvation. Cell samples for analysis were concentrated in I5-ml centrifuge tubes b y subjecting them to three 5-min centrifugations at IOOO×g. From o.3-1.2 cm 3 of packed amoebae were used for each determination (according to GRIFI~IN3 these volumes should contain between I - l O s and 6. lO s cells). T e t r a h y m e n a samples were prepared for analysis b y centrifuging pure cultures, washing twice in amoeba medium, and then proceeding as for a m o e b a samples. The method of nucleotide analysis used was that of OSAWA et al. 4. lJltravioletabsorption measurements were carried out in a Zeiss spectrophotometer (Model PMQII). Complete spectra were obtained on the pooled peaks after removal of the formic acid in vacuum. The samples ranged in absorbancy at the absorption peak for adenylic acid from o.5oo-1.5oo for amoeba and from 3.00-4.o0 for tetrahymena. The feeding process in A. proteus consists of the ingestion of the living food organism, its enclosure in a food vacuole and its subsequent digestion. If the composition of the RNA of t h e food organism differs significantly from that of the amoeba then the fate of the bulk of this RNA can be followed and the time after ingestion at which its presence is no longer detectable can be established. From this point in starvation on, the observed base composition should represent the composition of the RNA of the amoeba alone. The results of the present analysis, summarized in Table I, indicate t h a t the food TABLE
I
BASE COMPOSITION OF R N A FROM d . proteus AND T. pyri/ormis A. proteus Mole (%)*
T. pyrilormis Freshly ]ed
Ado Gua Cyd Urd
Starved 4 days
Starved
xo days
3o.3~o.3 2o.6~o. 7 22.2io.2 26.7~o. 4
23.7~o. 4 28.2±0.4 26.7ii.I 21.7±1.o
2o.5~I. 5 28.0±0.4 3o.7±1.3 2o.3~o.3
2o.6±1.4 27.2±0.8" 29.5±1.7 22-5~o.8
i.ii
i.oi
I.O 3
i,oo
AT
o.75
1.2o
i .29
1.3o
Purine Pyrimidine
i .0 4
I.o 7
o.9I
0.92
6-Amino 6-Oxo
GC
* T h e s e d a t a are t h e a v e r a g e s of d u p l i c a t e s a m p l e s . Biochim. Biophys. Acta, 55 (i962) 4 1 8 - 4 2 o
420
PRELIMINARY NOTES
organism used, T. pyri/ormis, has an RNA complement whose composition is distinctly different from that of A. proteus. The data are in good agreement with those of SCHERBAUM5 for this strain of tetrahymena. The presence of the foreign RNA is evident in the analysis of A. proteus immediately after feeding, as indicated by the lower GC/AU ratio and b y the high purine/pyrimidine ratio. The difference is absent 4 days later. I t follows that within the limits of precision of our analysis, t e t r a h y m e n a RNA is no longer detectable in the amoeba four days after its introduction. Unpublished experiments performed in our laboratory with t e t r a h y m e n a whose RNA was heavily labeled show that this RNA is not rejected b y the amoeba as such. It is most probable that the RNA of the food organism loses its characteristic identity b y degradation to at least the nucleotide level. (While even cursory examination suggests that amoebae feeding on t e t r a h y m e n a for extended periods do not lose their identity and begin to resemble their prey, it is reassuring to find that this conclusion is not contradicted at the molecular level.) The distribution of RNA-bound Azure B stain in the starved amoebae of the cultures used for chemical analysis was found to be predominantly homogeneous. This observation not only confirms the absence of undigested masses of t e t r a h y m e n a RNA, but further indicates that the possible contribution of any RNA-containing bodies of light-microscope dimension (i.e. yeast or bacteria contained in vacuoles, or symbiotic microorganisms) is negligible in comparison to the mass of RNA from the nucleus and cytoplasm of the amoeba. In summary, the results indicate that the A. proteus RNA is of the G-C type, that of T. pyri/ormis of the A - U type, and that the tetrahymenal RNA is assimilated b y the amoeba before the fourth day after ingestion of the living ciliate. This work was supported by grant No. RG-6317, National Institutes of Health, and by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation.
Department o/ Zoology, University o/ Wisconsin, Madison, Wisc. (U.S.A.) 1 2 3 4 6
J. E. CUMMINS W. PLAUT
A. I. COHEN, J. Biophys. Biochem. Cytol., 3 (1957) 923. D. PRESCOTT AND T. JAMES, Exptl. Cell Research, 8 (1955) 256. j . L. GRIFFIN, Exptl. Cell Research, 21 (196o) 17o. S. OSAWA, K. TAKATA AND Y. HOTTA, Biochim. Biophys. Acta, 28 (1958) 229. O. SCHERBAUM, Exptl. Cell Research, 13 (1957) 24.
Received December I l t h , 1961
Biochim. Biophys. Acta, 55 (1962) 418-42o