Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 642-645, February 1975
Induction by RNA of Inositol Independence in Neurospora crassa (mRNA/gene transformation)
N. C. MISHRA*T, M. C. NIUt, AND E. L. TATUM* * The Rockefeller University, New York, N.Y. 10021; and t Department of Biology, Temple University, Philadelphia, Pennsylvania 19122
Contributed by E. L. Tatum, December 9, 1974 The effect of purified wild-type RNA (allo-ABSTRACT RNA) on genetic reversion of inositol-requiring mutant 89601 of Neurospora crassa is described. The mutant (inos-) strain, on treatment with the wild-type RNA preparation, was found to revert to wild type (inos+) in significant numbers. RNA from the mutant (iso-RNA) and allo-RNA digested by RNase were ineffective in causing genetic reversion at the inositol locus. The allo-RNA-induced revertants were stable and showed a Mendelian transmission of the inos+ character.
supplement. After a heat treatment at 600 for 30 min, the medium containing the ascospores was distributed in sterile disposable petri plates (25 ml per plate). These plates were incubated at 250 for about 70 hr and then the growing colonies were counted. The inositol-independent revertant strains were examined for heterokaryosis (inos+/inos-) by plating equal aliquots of a suspension of conidia or mycelial fragments on sorbose-agar medium with and without inositol supplement. For a heterokaryotic culture, the number of colonies growing on inositolsupplemented medium would exceed significantly the number growing on the minimal medium.
The role of DNA in genetic transformation is well known (1). Recently, such a role for RNA has been suggested by work with several biological systems. The acquisition of a drugresistant character by sensitive bacteria after treatment with purified RNA preparations from resistant strains has been described in pneumococcus (2), Bacillus subtilis (3), and Escherichia coli (4). The transfer of other genetic characters mediated by purified RNA preparations has also been reported (5, 6). In higher organisms the development of specifictissues in response to exogenous RNA during organ culture has been described (7, 8). The possible role of RNA in transfer of genetic information is supported by the discovery of the enzyme, reverse transcriptase (RNA-dependent DNA polymerase) (9, 10), which can transcribe an RNA template into DNA. In this paper we describe the effect of exogenous RNA on genetic reversion at the inositol locus of Neurospora crassa. A preliminary account of this work has been presented (11).
RNA Preparation. The RNA was prepared by phenol extraction following standard procedures. The wild-type (inos+) and the mutant (inos-) strains of Neurospora were grown in carboys (20 liters capacity) containing 15 liters of Vogel's growth medium with or without inositol. Seventy grams (wet weight) of an actively growing culture (20-hr-old) were homogenized with about 150 g of sea sand and then suspended in 210 ml of 0.1 M Tris- HCl buffer (pH 8.0) containing sodium dodecyl sulfate (0.5%), NaCl (0.75 M), and ethylenediamine tetraacetic acid (0.025 M). This was centrifuged at 650 X g for 30 min and the supernatant (200 ml) was saved. To the supernatant were added 200 ml of 0.1 M Tris HCl buffer (pH 8.0) saturated with phenol. The solution was then homogenized in a Waring blender for 30 see at high speed and for another 30 see at low speed. The homogenate was then centrifuged at 12,100 X g for 15 min. The upper aqueous phase was saved and the lower phase was centrifuged after the addition of 1/2 volume of 0.9% saline. The upper aqueous phase was again saved. The two aqueous portions containing the RNA were combined and then reextracted with phenol; the upper aqueous phase was saved and RNA was precipitated by addition of 2 volumes of 95% alcohol. The RNA precipitate was collected by centrifugation and then dissolved in saline; afterwards, precipitation with alcohol was repeated four to five times. Preparation of Polyadenylate-Rich RNA Fraction. A large number of eukaryotic organisms are known to contain polyadenine sequences in their messenger RNA (17) and the mRNA from a variety of higher organisms has been prepared by absorption on nitrocellulose filters at a high-salt concentration and by subsequent elution with an appropriate buffer (18, 19). The Neurospora polyadenylate-rich RNA fraction was prepared by similar methods, as follows. The Neurospora bulk RNA preparation after phenol extraction and alcoholic reprecipitation was dissolved in 400 ml of 100 mM Tris- HCI
MATERIALS AND METHODS Strains. The Neurospora strains used were the wild-type
(RL3-8A, RL21a) strains, the inositol-requiring morphological mutant strain (R2506-8-12, inos-rg-A) and the inositolrequiring strain 89601. These strains were obtained from the Rockefeller University collection and were maintained on minimal medium (12) supplemented with inositol. Chemicals. Deoxyribonuclease I (RNase-free) and ribonuclease were obtained from Worthington Biochemical Co. Other chemicals were reagent grade. Nitrocellulose membranes were obtained from the Millipore Co. Genetic Analysis. The genetic crosses were made on synthetic crossing medium (13). The genetic crosses and the heterokaryon analysis were performed by standard methods as described earlier (14). A large sample of ascospores was analyzed by methods described by Giles (15). Equal aliquots of ascospore suspension were added to liquified agar sorbose medium (16) with or without inositol t Present address: Department of Biology, University of South Carolina, Columbia, S.C. 29208. 642
Proc. Nat. Acad. Sci. USA 72
RNA-Induced Inositol Independence in Neurospora
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TABLE 2. Effect of iso-RNA (inos -) on the genetic reversion of the inositolless strain of Neurospora crassa
TABLE 1. Effect of allo-RNA (inos+) on genetic reversion of the inositolless (inos-) locus in Neurospora crassa
Total number of Total number of
ColTreatment
1. Allo-RNA (inos+)b 2. Saline (without RNA) 3. RNase-treated alloRNA
Experiments
Treatment
ments
Colonies (X 10-)
Iso-RNA (inos-)b Saline (without RNA)
9 (0)c 9 (0)'c
54 61
Revertants
Freonies (X 106) Total quency0
12 (11)c
78.6
25
0.31
20 (1)c
160.0
2
0.01
8 (0)°
57.0
0
0
a Frequency expressed as per million. The polyadenylate-rich RNA fraction from the wild-type strain (inos +) (i.e., allo-RNA) was dissolved in saline and then added to the mutant recipient culture (inos. ) in a final concentration of 50.ug/ml. e Number of experiments with revertants in parentheses. b
buffer (pH 7.6) with 500 mM KCl and 1 mM MgCl2 and then filtered through a nitrocellulose membrane (Millipore, 47 mm diameter), (10 ml of RNA preparation per filter). Each membrane was washed with an equal volume of the same Tris buffer. After washing, the nitrocellulose membranes were collected and cut into small pieces; the absorbed polyadenylate-rich RNA was eluted with 10 mM Tris buffer (pH 9.0) with 0.5% sodium dodecyl sulfate using 3 ml per filter. The membrane pieces were then washed with same Tris buffer (10 mM, pH 9.0) but without the detergent. The eluates and the washings containing polyadenylate-rich RNA were combined and chilled and then centrifuged for 15 min at 480 X g to remove the detergent. NaCl to a concentration of 200 mM was added to the clear supernatant solution obtained after centrifugation. Polyadenylate-rich RNA was precipitated with 2 volumes of 95% alcohol. After chilling overnight, RNA was collected by centrifugation. The polyadenylate-rich RNA, presumably the mRNA fraction, so obtained was treated with Worthington DNase I (RNase-free). Preparations so obtained were free of detectable amounts of DNA and protein and were then used for the reversion experiments. Allo- and Iso-RNA Preparations. The RNA preparation from the wild-type strain (RL3-8A) was designated as alloRNA and the comparable RNA preparation from the inositolrequiring mutant strain (R2506-8-12) was called iso-RNA. DNA and RNA were chemically determined by diphenylamine and orcinol reactions, respectively (20), and protein was determined by the method of Lowry et al. (21). Nucleic acids and proteins were also routinely determined spectrophotometrically. RNA was dissolved in saline solution and kept at 5° before use. Enzyme Digestion. Different RNA preparations were treated with DNase I or with RNase (ribonuclease) following methods described by Marmur (22). RNA Treatment of the Neurospora Culture. The role of RNA in genetic transformation was determined by its ability to direct the reversion of inositolless allele (89601) in Neurospora. The rationale for using this particular inositolless mutant strain in the present study has been provided earlier (11). Typically, the mutant strain (inos-) was used as the recipient culture and was treated with either allo-RNA or iso-RNA.
Experi-
Revertants
FreTotal quency, 0 0
0 0
a Frequency expressed as per million. b The RNA preparation from the mutant strain (inos -) was dissolved in saline and then added to the recipient culture (inos-) in a final concentration of 50.ug/ml. e Number of experiments with revertants in parentheses.
The culture was then examined for reversion to inositol independence by testing its ability to grow on medium without inositol. The actively growing mutant (inos-, rg-A) culture was briefly homogenized (20 sec) in 20% sucrose in sodium phosphate buffer (0.2 M, pH 6.5). An aliquot (0.05 ml) of the mycelial suspension thus obtained was added to 50 ml Pyrex flasks containing 10 ml of Neurospora growth medium (12). The RNA preparation in saline solution was added so that the final concentration of RNA in the treated culture was 50 /Ag/ml. In control experiments the recipient culture received only saline (but no allo-RNA) solution. In some other control experiments, the recipient culture was treated with allo-RNA digested by RNase or with iso-RNA. The control and the allo-RNA-treated cultures were started in duplicate flasks from the same sample of the recipient mycelial suspension; thus these cultures were identical except for the RNA treatment. After 60 hr of growth, the mycelia from each duplicate set of the control or of the treated cultures were pooled and suspended in 5 ml of 20% sucrose in sodium phosphate buffer (0.2 M, pH 6.5). Each mycelial suspension was separately subjected to brief homogenization to break the hyphae into small fragments; these were then plated on minimal and on inositol-supplement medium. The inositol-independent colonies growing on minimal medium were scored as RNAinduced revertants. The number of colonies growing on the inositol-supplemented medium was counted and used to calculate the total number of colony-forming units plated. The induced inositol-independent revertant (inos+) colonies scored on minimal medium after RNA treatment were maintained on minimal medium and later used for genetic analysis in crosses with the inos- strain (89601a). RESULTS Effect of Allo-RNA. The data presented in Table 1 show that treatment with allo-RNA produced significant numbers of inositol-independent revertants as compared to-the control experiments without RNA treatment. With the allo-RNAtreated culture approximately 79 million colonies were examined and 25 revertants were obtained. Only two revertants appeared among 160 million colonies examined from the control experiment receiving no RNA (see Table 1). The data presented in Table 1 also show that among the experiments performed with allo-RNA a significant number (11 out of 12 experiments) resulted in the production of inos+ revertants, whereas in control experiments (without RNA treatment)
Genetics: Mishra et al.
644
Proc. Nat. Acad. Sci. USA 72 (1975)
TABLE 3. Genetic analysis by plating of the wild-type revertants (inos+) obtained with allo-RNA treatment No. of progeny
No. of progeny
Crossa
inos-
inos +
Crossa
inos-
inos +
1 2 3 4 5 6 7 8
712 306 118 483 871 402 1113 687
693 327 129 469 886 392 1149 669
9 10 11 12 13 14 15 16 17b
521 263 489 679 473 503 656 447 172
533 257 507 656 485 516 667 463 526
a Revertant (inos +) X
(inosj (89601a).
Inositol-independent revertant found (inos +/inos I (see text). b
to be heterokaryotic
only one of the 20 experiments produced revertants. In other control experiments no revertants were found to occur among a large number of experiments performed (see Tables 1 and 2). The different controls included those experiments in which the recipient- cultures were treated with saline alone or with RNase-digested allo-RNA or with iso-RNA; these results are presented in Tables 1 and 2. Effect of Enzyme Digestion. In order to determine whether or not the effect of allo-RNA on genetic reversion was specific, the RNA preparation was treated with the enzyme RNase and was then used in treatment of the recipient culture. In such studies with enzymatically digested allo-RNA no wildtype revertants were produced (see Table 1). However, the RNA preparations were routinely subjected to enzyme digestion by DNase I (RNase free) before use and were effective in inducing reversion of the inositolless strain (inos-) into the wild type (inos+).
Effect of Iso-RNA. It seems that the reversion of the inositolless strain (inos-) to inositol independence (inos+) was due to the transforming ability of the allo-RNA preparation carrying the wild-type (inos+) information. If this is true, then a similar RNA preparation from the mutant culture, which would be carrying mutant genetic information (inos-), should have no effect on the reversion of the recipient (inos-) culture. The recipient culture was, therefore, treated with an iso-RNA preparation (i.e., with its own RNA) and then examined for inositol-independent revertants. No wild-type revertants were obtained in such experiments. The data are presented in Table 2. Stability of Allo-RNA Induced Revertants. The inositolindependent revertants obtained after allo-RNA treatment were maintained on minimal medium. They appeared to be completely stable, since they have retained the inos+ character for over 2 years, and the addition of inositol did not affect their growth. Genetic Analysis. In order to determine the mode of inheritance of the allo-RNA-induced inositol independence character in Neurospora, an extensive genetic analysis of these revertants was carried out. The revertants (inos+) were crossed with the inositol-requiring strain 89601 (inos-) and the progeny were examined for growth on minimal medium
and on inositol-supplemented medium. Results of the random spore analysis of the crosses are presented in Table 3. In such crosses (except for the cross no. 17 in Table 3) the inos+ and inos- characters were found to be transmitted in the expected Mendelian ratio of 1:1. These crosses were also examined by tetrad analysis. Ten complete asci (one ascus/perithecium) were analyzed from each cross and these were found to have 4 inos+ and 4 inos- spores in each ascus (except the cross no. 17). Asci from cross no. 17 (Table 3) were found to be of two kinds; these have either 4 inos+ and 4 inos- spores, or 8 inos- spores, in each ascus, suggesting the heterokaryotic (inos+/inos-) nature of the revertant parent involved in this cross (no. 17 Table 3). This revertant strain was, therefore, examined for heterokaryosis at the inositol locus (inos+/ inos-). Equal aliquots of the mycelial fragments were plated on minimal medium and also on the inositol-supplemented medium; the numbers of colonies growing on these two kinds of media were 29 and 105, respectively. This provided the evidence that this strain was indeed heterokaryotic. Except for this revertant (no. 17), the others, on similar analysis, were found to give equal numbers of colonies on the minimal and on the inositol-supplemented media. They were, therefore, homokaryotic for the inos+ allele. DISCUSSION The data presented in this paper provide evidence for the
RNA-induced reversion of the inositol locus (inos-) in Neurospora. The specificity of the RNA effect is suggested by the fact that the preparation of the mutant's own RNA was not effective in inducing reversion at this locus and also that RNase treatment abolished the activity of the RNA preparation, whereas DNase treatment did not seem to have any adverse effect on its activity. Our method of RNA (polyadenylate-rich fraction) preparation and the fact that this RNA preparation showed a negative diphenylamine test suggest that our RNA preparation was not a DNA -RNA hybrid. The stability of the RNA-induced revertants suggests that the genetic information carried by exogenous RNA was capable of replication, transcription, and translation in the recipient cells. That these RNA-induced revertants transmitted their induced character [i.e., inositol independence (inos+)] to their sexual progeny in Mendelian ratios is in contrast to the non-Mendelian mode of inheritance of some DNA-induced revertants (14). This suggests that the genetic information carried by exogenous RNA is readily transferred to the recipient DNA and thus would appear to implicate the enzyme reverse transcriptase. The low frequency of allo-RNA-induced reversion observed during the present study (see Tables 1 and 2) seems, indeed, to be significant in view of the fact that no spontaneous reversion or an exceedingly low induced reversion frequency has been found for this particular inositolless allele (89601),
by Giles (15). The authors wish to thank Miss Anne Hamill for her expert technical help. This work was supported in part by a grant from the National Institutes of Health (GM-16224) to E.L.T. 1. Hotchkiss, R. D.
(1971) "Introductory remarks," in In-
formative Molecules in Biological Systems, ed. Ledoux, L. H. (North-Holland Publ. Co., Amsterdam), pp. 1-3. 2. Evans, A. (1964) "Introduction of specific drug resistance
Proc. Nat. Acad. Sci. USA 72
3. 4.
5.
6.
7. 8. 9. 10. 11.
12.
(1975)
properties by purified RNA containing fractions from pneumococcus," Proc. Nat. Acad. Sci. USA 52, 1442-1449. Shen, S. C., Hong, M., Cai, R., Chen, W. & Chang, W. (1962) Sci. Sinica 11, (2) 233. Quoted by Cheng, T. & Doi, R. H. (1968) Prog. Nuci. Acid Res. Mol. Biol. 8, 335. Beljanski, M., Beljanski, M. S. &Bourgarel, P. (1971) "ARN Transformants porteurs de caractkires h6r6ditaires chez Escherichia coli showdomycin r6sistant," C.R.H. Acad. Sci. Ser D. 272, 2107-2111. Beljanski, M., Beljanski, M. S., Manigault, P. & Bourgarel, P. (1972) "Transformation of Agrobacterium tumefaciens into non-oncogenic species by an Escherichia coli RNA," Proc. Nat. Acad. Sci. USA 69, 191-195. Beljanski, M., Aaron-Da-Cunha, M. I., Beljanski, M. S., Manigault, P., & Bourgarel, P. (1974) "Isolation of the tumor-inducing RNA from oncogenic and nononcogenic Agrobacterium tumefaciens," Proc. Nat. Acad. Sci. USA 71, 1585-1589. Segal, S. J., Davidson, 0. W. & Wada, K. (1965) "Role of RNA in the regulatory action of estrogen," Proc. Nat. Acad. Sci. USA 54, 782-787. Niu, M. C. (1958) "Thymus ribonucleic acid and embryonic differentiation," Proc. Nat. Acad. Sci. USA 44, 1264-1274. Temin, H. M. & Mizutani, S. (1970) "RNA-dependent DNA polymerase in virions of Rous sarcoma virus," Nature 226, 1211-1213. Baltimore, D. (1970) "RNA dependent DNA polymerase in virions of RNA tumor viruses," Nature 226, 1209-1211. Mishra, N. C., Szabo, G. & Tatum, E. L. (1973) "Nucleic acid induced genetic changes in Neurospora," in The Role of RNA in Reproduction and Development, eds. Niu, M. C. & Segal, S. J. (North-Holland Publ. Co., Amsterdam), pp. 259-268. Vogel, H. J. (1964) "Distribution of lysine pathways among
RNA-Induced Inositol Independence in Neurospora
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fungi: Evolutionary implications," Amer. Natur. 98,
435-446. 13. Westergaard, M. & Mitchell, H. K. (1947) "Neurospora V. A synthetic medium favoring sexual reproduction," Amer. J. Bot. 34, 573-577. 14. Mishra, N. C. & Tatum, E. L. (1973) "Non-Mendelian inheritance of DNA-induced inositol independence in Neurospora," Proc. Nat. Acad. Sci. USA 70, 3875-3879. 15. Giles, N. H., Jr. (1951) "Studies on the mechanism of reversion in biochemical mutants of Neurospora crassa," Cold Spring Harbor Symp. Quant. Biol. 16, 283-319. 16. Tatum, E. L., Barratt, R. W. & Cutter, V. M. (1949) "Chemical induction of colonial paramorphs in Neurospora and Syncephalastrum," Science 109, 509-511. 17. Darnell, J. E., Jelinek, W. R. & Molloy, G. R. (1973) "Biogenesis of mRNA; Genetic regulation in mammalian cells," Science 181, 1215-1221. 18. Brawerman, G., Mendicki, J. & Lee, S. Y. (1972) "A procedure for isolation of mammalian messenger ribonucleic acid," Biochemistry 11, 637-641. 19. Rosenfeld, G. C., Comstock, J. P., Means, A. R. & O'Malley, B. C. (1972) "A rapid method for the isolation and partial purification of specific eukaryotic messenger RNA's," Biochem. Biophys. Res. Commun. 47, 387-392. 20. Schneider, W. C. (1953) "Determination of nucleic acids in tissue by pentose analysis," in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 3, pp. 680-683. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & R~ndall, R. J. (1951) "Protein measurement with the Folin phenol reagent," J. Biol. Chem. 193, 265-275. 22. Marmur, J. (1961) "A procedure for isolation of deoxyribonucleic acids from microorganisms," J. Mol. Biol. 3, 208-218.
Induction by RNA of Inositol Independence in Neurospora crassa (mRNA/gene transformation)
N. C. MISHRA*T, M. C. NIUt, AND E. L. TATUM* * The Rockefeller University, New York, N.Y. 10021; and t Department of Biology, Temple University, Philadelphia, Pennsylvania 19122
Contributed by E. L. Tatum, December 9, 1974 The effect of purified wild-type RNA (allo-ABSTRACT RNA) on genetic reversion of inositol-requiring mutant 89601 of Neurospora crassa is described. The mutant (inos-) strain, on treatment with the wild-type RNA preparation, was found to revert to wild type (inos+) in significant numbers. RNA from the mutant (iso-RNA) and allo-RNA digested by RNase were ineffective in causing genetic reversion at the inositol locus. The allo-RNA-induced revertants were stable and showed a Mendelian transmission of the inos+ character.
supplement. After a heat treatment at 600 for 30 min, the medium containing the ascospores was distributed in sterile disposable petri plates (25 ml per plate). These plates were incubated at 250 for about 70 hr and then the growing colonies were counted. The inositol-independent revertant strains were examined for heterokaryosis (inos+/inos-) by plating equal aliquots of a suspension of conidia or mycelial fragments on sorbose-agar medium with and without inositol supplement. For a heterokaryotic culture, the number of colonies growing on inositolsupplemented medium would exceed significantly the number growing on the minimal medium.
The role of DNA in genetic transformation is well known (1). Recently, such a role for RNA has been suggested by work with several biological systems. The acquisition of a drugresistant character by sensitive bacteria after treatment with purified RNA preparations from resistant strains has been described in pneumococcus (2), Bacillus subtilis (3), and Escherichia coli (4). The transfer of other genetic characters mediated by purified RNA preparations has also been reported (5, 6). In higher organisms the development of specifictissues in response to exogenous RNA during organ culture has been described (7, 8). The possible role of RNA in transfer of genetic information is supported by the discovery of the enzyme, reverse transcriptase (RNA-dependent DNA polymerase) (9, 10), which can transcribe an RNA template into DNA. In this paper we describe the effect of exogenous RNA on genetic reversion at the inositol locus of Neurospora crassa. A preliminary account of this work has been presented (11).
RNA Preparation. The RNA was prepared by phenol extraction following standard procedures. The wild-type (inos+) and the mutant (inos-) strains of Neurospora were grown in carboys (20 liters capacity) containing 15 liters of Vogel's growth medium with or without inositol. Seventy grams (wet weight) of an actively growing culture (20-hr-old) were homogenized with about 150 g of sea sand and then suspended in 210 ml of 0.1 M Tris- HCl buffer (pH 8.0) containing sodium dodecyl sulfate (0.5%), NaCl (0.75 M), and ethylenediamine tetraacetic acid (0.025 M). This was centrifuged at 650 X g for 30 min and the supernatant (200 ml) was saved. To the supernatant were added 200 ml of 0.1 M Tris HCl buffer (pH 8.0) saturated with phenol. The solution was then homogenized in a Waring blender for 30 see at high speed and for another 30 see at low speed. The homogenate was then centrifuged at 12,100 X g for 15 min. The upper aqueous phase was saved and the lower phase was centrifuged after the addition of 1/2 volume of 0.9% saline. The upper aqueous phase was again saved. The two aqueous portions containing the RNA were combined and then reextracted with phenol; the upper aqueous phase was saved and RNA was precipitated by addition of 2 volumes of 95% alcohol. The RNA precipitate was collected by centrifugation and then dissolved in saline; afterwards, precipitation with alcohol was repeated four to five times. Preparation of Polyadenylate-Rich RNA Fraction. A large number of eukaryotic organisms are known to contain polyadenine sequences in their messenger RNA (17) and the mRNA from a variety of higher organisms has been prepared by absorption on nitrocellulose filters at a high-salt concentration and by subsequent elution with an appropriate buffer (18, 19). The Neurospora polyadenylate-rich RNA fraction was prepared by similar methods, as follows. The Neurospora bulk RNA preparation after phenol extraction and alcoholic reprecipitation was dissolved in 400 ml of 100 mM Tris- HCI
MATERIALS AND METHODS Strains. The Neurospora strains used were the wild-type
(RL3-8A, RL21a) strains, the inositol-requiring morphological mutant strain (R2506-8-12, inos-rg-A) and the inositolrequiring strain 89601. These strains were obtained from the Rockefeller University collection and were maintained on minimal medium (12) supplemented with inositol. Chemicals. Deoxyribonuclease I (RNase-free) and ribonuclease were obtained from Worthington Biochemical Co. Other chemicals were reagent grade. Nitrocellulose membranes were obtained from the Millipore Co. Genetic Analysis. The genetic crosses were made on synthetic crossing medium (13). The genetic crosses and the heterokaryon analysis were performed by standard methods as described earlier (14). A large sample of ascospores was analyzed by methods described by Giles (15). Equal aliquots of ascospore suspension were added to liquified agar sorbose medium (16) with or without inositol t Present address: Department of Biology, University of South Carolina, Columbia, S.C. 29208. 642
Proc. Nat. Acad. Sci. USA 72
RNA-Induced Inositol Independence in Neurospora
(1975)
643
TABLE 2. Effect of iso-RNA (inos -) on the genetic reversion of the inositolless strain of Neurospora crassa
TABLE 1. Effect of allo-RNA (inos+) on genetic reversion of the inositolless (inos-) locus in Neurospora crassa
Total number of Total number of
ColTreatment
1. Allo-RNA (inos+)b 2. Saline (without RNA) 3. RNase-treated alloRNA
Experiments
Treatment
ments
Colonies (X 10-)
Iso-RNA (inos-)b Saline (without RNA)
9 (0)c 9 (0)'c
54 61
Revertants
Freonies (X 106) Total quency0
12 (11)c
78.6
25
0.31
20 (1)c
160.0
2
0.01
8 (0)°
57.0
0
0
a Frequency expressed as per million. The polyadenylate-rich RNA fraction from the wild-type strain (inos +) (i.e., allo-RNA) was dissolved in saline and then added to the mutant recipient culture (inos. ) in a final concentration of 50.ug/ml. e Number of experiments with revertants in parentheses. b
buffer (pH 7.6) with 500 mM KCl and 1 mM MgCl2 and then filtered through a nitrocellulose membrane (Millipore, 47 mm diameter), (10 ml of RNA preparation per filter). Each membrane was washed with an equal volume of the same Tris buffer. After washing, the nitrocellulose membranes were collected and cut into small pieces; the absorbed polyadenylate-rich RNA was eluted with 10 mM Tris buffer (pH 9.0) with 0.5% sodium dodecyl sulfate using 3 ml per filter. The membrane pieces were then washed with same Tris buffer (10 mM, pH 9.0) but without the detergent. The eluates and the washings containing polyadenylate-rich RNA were combined and chilled and then centrifuged for 15 min at 480 X g to remove the detergent. NaCl to a concentration of 200 mM was added to the clear supernatant solution obtained after centrifugation. Polyadenylate-rich RNA was precipitated with 2 volumes of 95% alcohol. After chilling overnight, RNA was collected by centrifugation. The polyadenylate-rich RNA, presumably the mRNA fraction, so obtained was treated with Worthington DNase I (RNase-free). Preparations so obtained were free of detectable amounts of DNA and protein and were then used for the reversion experiments. Allo- and Iso-RNA Preparations. The RNA preparation from the wild-type strain (RL3-8A) was designated as alloRNA and the comparable RNA preparation from the inositolrequiring mutant strain (R2506-8-12) was called iso-RNA. DNA and RNA were chemically determined by diphenylamine and orcinol reactions, respectively (20), and protein was determined by the method of Lowry et al. (21). Nucleic acids and proteins were also routinely determined spectrophotometrically. RNA was dissolved in saline solution and kept at 5° before use. Enzyme Digestion. Different RNA preparations were treated with DNase I or with RNase (ribonuclease) following methods described by Marmur (22). RNA Treatment of the Neurospora Culture. The role of RNA in genetic transformation was determined by its ability to direct the reversion of inositolless allele (89601) in Neurospora. The rationale for using this particular inositolless mutant strain in the present study has been provided earlier (11). Typically, the mutant strain (inos-) was used as the recipient culture and was treated with either allo-RNA or iso-RNA.
Experi-
Revertants
FreTotal quency, 0 0
0 0
a Frequency expressed as per million. b The RNA preparation from the mutant strain (inos -) was dissolved in saline and then added to the recipient culture (inos-) in a final concentration of 50.ug/ml. e Number of experiments with revertants in parentheses.
The culture was then examined for reversion to inositol independence by testing its ability to grow on medium without inositol. The actively growing mutant (inos-, rg-A) culture was briefly homogenized (20 sec) in 20% sucrose in sodium phosphate buffer (0.2 M, pH 6.5). An aliquot (0.05 ml) of the mycelial suspension thus obtained was added to 50 ml Pyrex flasks containing 10 ml of Neurospora growth medium (12). The RNA preparation in saline solution was added so that the final concentration of RNA in the treated culture was 50 /Ag/ml. In control experiments the recipient culture received only saline (but no allo-RNA) solution. In some other control experiments, the recipient culture was treated with allo-RNA digested by RNase or with iso-RNA. The control and the allo-RNA-treated cultures were started in duplicate flasks from the same sample of the recipient mycelial suspension; thus these cultures were identical except for the RNA treatment. After 60 hr of growth, the mycelia from each duplicate set of the control or of the treated cultures were pooled and suspended in 5 ml of 20% sucrose in sodium phosphate buffer (0.2 M, pH 6.5). Each mycelial suspension was separately subjected to brief homogenization to break the hyphae into small fragments; these were then plated on minimal and on inositol-supplement medium. The inositol-independent colonies growing on minimal medium were scored as RNAinduced revertants. The number of colonies growing on the inositol-supplemented medium was counted and used to calculate the total number of colony-forming units plated. The induced inositol-independent revertant (inos+) colonies scored on minimal medium after RNA treatment were maintained on minimal medium and later used for genetic analysis in crosses with the inos- strain (89601a). RESULTS Effect of Allo-RNA. The data presented in Table 1 show that treatment with allo-RNA produced significant numbers of inositol-independent revertants as compared to-the control experiments without RNA treatment. With the allo-RNAtreated culture approximately 79 million colonies were examined and 25 revertants were obtained. Only two revertants appeared among 160 million colonies examined from the control experiment receiving no RNA (see Table 1). The data presented in Table 1 also show that among the experiments performed with allo-RNA a significant number (11 out of 12 experiments) resulted in the production of inos+ revertants, whereas in control experiments (without RNA treatment)
Genetics: Mishra et al.
644
Proc. Nat. Acad. Sci. USA 72 (1975)
TABLE 3. Genetic analysis by plating of the wild-type revertants (inos+) obtained with allo-RNA treatment No. of progeny
No. of progeny
Crossa
inos-
inos +
Crossa
inos-
inos +
1 2 3 4 5 6 7 8
712 306 118 483 871 402 1113 687
693 327 129 469 886 392 1149 669
9 10 11 12 13 14 15 16 17b
521 263 489 679 473 503 656 447 172
533 257 507 656 485 516 667 463 526
a Revertant (inos +) X
(inosj (89601a).
Inositol-independent revertant found (inos +/inos I (see text). b
to be heterokaryotic
only one of the 20 experiments produced revertants. In other control experiments no revertants were found to occur among a large number of experiments performed (see Tables 1 and 2). The different controls included those experiments in which the recipient- cultures were treated with saline alone or with RNase-digested allo-RNA or with iso-RNA; these results are presented in Tables 1 and 2. Effect of Enzyme Digestion. In order to determine whether or not the effect of allo-RNA on genetic reversion was specific, the RNA preparation was treated with the enzyme RNase and was then used in treatment of the recipient culture. In such studies with enzymatically digested allo-RNA no wildtype revertants were produced (see Table 1). However, the RNA preparations were routinely subjected to enzyme digestion by DNase I (RNase free) before use and were effective in inducing reversion of the inositolless strain (inos-) into the wild type (inos+).
Effect of Iso-RNA. It seems that the reversion of the inositolless strain (inos-) to inositol independence (inos+) was due to the transforming ability of the allo-RNA preparation carrying the wild-type (inos+) information. If this is true, then a similar RNA preparation from the mutant culture, which would be carrying mutant genetic information (inos-), should have no effect on the reversion of the recipient (inos-) culture. The recipient culture was, therefore, treated with an iso-RNA preparation (i.e., with its own RNA) and then examined for inositol-independent revertants. No wild-type revertants were obtained in such experiments. The data are presented in Table 2. Stability of Allo-RNA Induced Revertants. The inositolindependent revertants obtained after allo-RNA treatment were maintained on minimal medium. They appeared to be completely stable, since they have retained the inos+ character for over 2 years, and the addition of inositol did not affect their growth. Genetic Analysis. In order to determine the mode of inheritance of the allo-RNA-induced inositol independence character in Neurospora, an extensive genetic analysis of these revertants was carried out. The revertants (inos+) were crossed with the inositol-requiring strain 89601 (inos-) and the progeny were examined for growth on minimal medium
and on inositol-supplemented medium. Results of the random spore analysis of the crosses are presented in Table 3. In such crosses (except for the cross no. 17 in Table 3) the inos+ and inos- characters were found to be transmitted in the expected Mendelian ratio of 1:1. These crosses were also examined by tetrad analysis. Ten complete asci (one ascus/perithecium) were analyzed from each cross and these were found to have 4 inos+ and 4 inos- spores in each ascus (except the cross no. 17). Asci from cross no. 17 (Table 3) were found to be of two kinds; these have either 4 inos+ and 4 inos- spores, or 8 inos- spores, in each ascus, suggesting the heterokaryotic (inos+/inos-) nature of the revertant parent involved in this cross (no. 17 Table 3). This revertant strain was, therefore, examined for heterokaryosis at the inositol locus (inos+/ inos-). Equal aliquots of the mycelial fragments were plated on minimal medium and also on the inositol-supplemented medium; the numbers of colonies growing on these two kinds of media were 29 and 105, respectively. This provided the evidence that this strain was indeed heterokaryotic. Except for this revertant (no. 17), the others, on similar analysis, were found to give equal numbers of colonies on the minimal and on the inositol-supplemented media. They were, therefore, homokaryotic for the inos+ allele. DISCUSSION The data presented in this paper provide evidence for the
RNA-induced reversion of the inositol locus (inos-) in Neurospora. The specificity of the RNA effect is suggested by the fact that the preparation of the mutant's own RNA was not effective in inducing reversion at this locus and also that RNase treatment abolished the activity of the RNA preparation, whereas DNase treatment did not seem to have any adverse effect on its activity. Our method of RNA (polyadenylate-rich fraction) preparation and the fact that this RNA preparation showed a negative diphenylamine test suggest that our RNA preparation was not a DNA -RNA hybrid. The stability of the RNA-induced revertants suggests that the genetic information carried by exogenous RNA was capable of replication, transcription, and translation in the recipient cells. That these RNA-induced revertants transmitted their induced character [i.e., inositol independence (inos+)] to their sexual progeny in Mendelian ratios is in contrast to the non-Mendelian mode of inheritance of some DNA-induced revertants (14). This suggests that the genetic information carried by exogenous RNA is readily transferred to the recipient DNA and thus would appear to implicate the enzyme reverse transcriptase. The low frequency of allo-RNA-induced reversion observed during the present study (see Tables 1 and 2) seems, indeed, to be significant in view of the fact that no spontaneous reversion or an exceedingly low induced reversion frequency has been found for this particular inositolless allele (89601),
by Giles (15). The authors wish to thank Miss Anne Hamill for her expert technical help. This work was supported in part by a grant from the National Institutes of Health (GM-16224) to E.L.T. 1. Hotchkiss, R. D.
(1971) "Introductory remarks," in In-
formative Molecules in Biological Systems, ed. Ledoux, L. H. (North-Holland Publ. Co., Amsterdam), pp. 1-3. 2. Evans, A. (1964) "Introduction of specific drug resistance
Proc. Nat. Acad. Sci. USA 72
3. 4.
5.
6.
7. 8. 9. 10. 11.
12.
(1975)
properties by purified RNA containing fractions from pneumococcus," Proc. Nat. Acad. Sci. USA 52, 1442-1449. Shen, S. C., Hong, M., Cai, R., Chen, W. & Chang, W. (1962) Sci. Sinica 11, (2) 233. Quoted by Cheng, T. & Doi, R. H. (1968) Prog. Nuci. Acid Res. Mol. Biol. 8, 335. Beljanski, M., Beljanski, M. S. &Bourgarel, P. (1971) "ARN Transformants porteurs de caractkires h6r6ditaires chez Escherichia coli showdomycin r6sistant," C.R.H. Acad. Sci. Ser D. 272, 2107-2111. Beljanski, M., Beljanski, M. S., Manigault, P. & Bourgarel, P. (1972) "Transformation of Agrobacterium tumefaciens into non-oncogenic species by an Escherichia coli RNA," Proc. Nat. Acad. Sci. USA 69, 191-195. Beljanski, M., Aaron-Da-Cunha, M. I., Beljanski, M. S., Manigault, P., & Bourgarel, P. (1974) "Isolation of the tumor-inducing RNA from oncogenic and nononcogenic Agrobacterium tumefaciens," Proc. Nat. Acad. Sci. USA 71, 1585-1589. Segal, S. J., Davidson, 0. W. & Wada, K. (1965) "Role of RNA in the regulatory action of estrogen," Proc. Nat. Acad. Sci. USA 54, 782-787. Niu, M. C. (1958) "Thymus ribonucleic acid and embryonic differentiation," Proc. Nat. Acad. Sci. USA 44, 1264-1274. Temin, H. M. & Mizutani, S. (1970) "RNA-dependent DNA polymerase in virions of Rous sarcoma virus," Nature 226, 1211-1213. Baltimore, D. (1970) "RNA dependent DNA polymerase in virions of RNA tumor viruses," Nature 226, 1209-1211. Mishra, N. C., Szabo, G. & Tatum, E. L. (1973) "Nucleic acid induced genetic changes in Neurospora," in The Role of RNA in Reproduction and Development, eds. Niu, M. C. & Segal, S. J. (North-Holland Publ. Co., Amsterdam), pp. 259-268. Vogel, H. J. (1964) "Distribution of lysine pathways among
RNA-Induced Inositol Independence in Neurospora
645
fungi: Evolutionary implications," Amer. Natur. 98,
435-446. 13. Westergaard, M. & Mitchell, H. K. (1947) "Neurospora V. A synthetic medium favoring sexual reproduction," Amer. J. Bot. 34, 573-577. 14. Mishra, N. C. & Tatum, E. L. (1973) "Non-Mendelian inheritance of DNA-induced inositol independence in Neurospora," Proc. Nat. Acad. Sci. USA 70, 3875-3879. 15. Giles, N. H., Jr. (1951) "Studies on the mechanism of reversion in biochemical mutants of Neurospora crassa," Cold Spring Harbor Symp. Quant. Biol. 16, 283-319. 16. Tatum, E. L., Barratt, R. W. & Cutter, V. M. (1949) "Chemical induction of colonial paramorphs in Neurospora and Syncephalastrum," Science 109, 509-511. 17. Darnell, J. E., Jelinek, W. R. & Molloy, G. R. (1973) "Biogenesis of mRNA; Genetic regulation in mammalian cells," Science 181, 1215-1221. 18. Brawerman, G., Mendicki, J. & Lee, S. Y. (1972) "A procedure for isolation of mammalian messenger ribonucleic acid," Biochemistry 11, 637-641. 19. Rosenfeld, G. C., Comstock, J. P., Means, A. R. & O'Malley, B. C. (1972) "A rapid method for the isolation and partial purification of specific eukaryotic messenger RNA's," Biochem. Biophys. Res. Commun. 47, 387-392. 20. Schneider, W. C. (1953) "Determination of nucleic acids in tissue by pentose analysis," in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 3, pp. 680-683. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & R~ndall, R. J. (1951) "Protein measurement with the Folin phenol reagent," J. Biol. Chem. 193, 265-275. 22. Marmur, J. (1961) "A procedure for isolation of deoxyribonucleic acids from microorganisms," J. Mol. Biol. 3, 208-218.
