Replication and Expression of a Bacterial-Mitochondrial Hybrid

Proc. Nati Acad. Sci. USA Vol. 79, pp. 3641-3645, June 1982 Microbiology

Replication and expression of a bacterial-mitochondrial hybrid plasmid in the fungus Podospora anserina (Podospora transformation/hybrid shuttle vector/prokaryotic-eukaryotic gene expression) ULF STAHL, PAUL TUDZYNSKI, ULRICH KUCK, AND KARL ESSER Lehrstuhl fir Allgemeine Botanik, Ruhr-Universitat, D-4630 Bochum 1, Federal Republic of Germany Communicated by Kenneth V. Thimann, February 8, 1982

ABSTRACT Hybrid plasmids consisting of the bacterial plas- of genetic material? (iii) Further, will it be possible to use a pl mid pBR322 and plasmid-like DNA (pl DNA) sequences from the DNA sequence excised from juvenile mtDNA as a vector? This fungus Podospora anserina are not only able to replicate in Esch- would open a possibility for a different form of genetic engi- erichia coli but also in the fungus. This was proved by both bio- neering-one using a eukaryotic replicon of mitochondrial or- physical and biological evidence involving buoyant density pro- igin as a vector. files, DNADNA hybridization, and restriction analysis-all Data presented here show that genetic information from bac- confirming that pl DNA behaves as a true plasmid. During its terial DNA integrated with pl DNA can be expressed in trans- amplification in P. anserina, the hybrid plasmid does not lose its formed Podospora anserina. prokaryotic coding capacity as shown after retransfer and subse- quent cloning in E. coli P. ansermna is able to express both the eukaryotic and the prokaryotic genetic information of the hybrid MATERIAL AND METHODS plasmid because the occurrence ofsenescence and the production Strains. The wild strain s (American Type Culture Collection of .-lactamase could be shown in experiments involving specific 26003) and the double mutant grisealvivax (gr: grey asco- hybrid plasmids. In the same systems, it was possible to demon- spores; viv: aerial hyphae rhythmical) of Podospora anserina strate that a hybrid plasmid containing, instead of p1 DNA, a pl were used as recipients in DNA homologous region of native mtDNA also could function as transformation experiments (life span a true plasmid. This hybrid plasmid contained about 25% of the about 25 and >900 days, respectively). (For details on their genetic information ofpl DNA, which corresponds to about 6% of origin, ontogeny, and genetics, see K. Esser in ref. 9.) Escher- the genetic information of mtDNA. Thus, the data show that hy- ichia coli K-12 SF8 (recB21, recC22, lop-11, tonAl, thr-1, brid plasmids may be used to shuttle genetic information between leu-6, thi-1, lacYl, supE44, r-, m-) served as prokaryotic host. P. anserina and E. coli Hence, through the use ofa mtDNA repli- Plasmids. Single (pSP4, pSP24) or double (pSP17) copies of con, as evidenced by the pl DNA of P. anserina, another pathway pl DNA were integrated in the Sal I site of pBR322 (10). In in genetic engineering is established. pSP24 and pSP17, pl DNA is orientated the same; in pSP4, the orientation is inverted (details are in ref. 6). pKP402 is con- The senescence that occurs regularly in wild strains of the as- structed like pSP4 but contains the Sal I fragment 4 of mtDNA comycete Podospora anserina (1) is caused by an infective agent from P. anserina as a eukaryotic constituent (7). (2) identical with plasmid-like DNA (pl DNA) (3, 4). This DNA Methods. Culture conditions and media, isolation of DNA, species is not found in juvenile mycelia and has the following and the determination of buoyant densities were as described properties: contour length, 0.75 tkm; buoyant density, 1.699 g/ by Stahl et al. (6). Transformation procedures were carried out cm3; and molecular size, 2.4 kilobases (kb). Confirmative ob- as described for P. anserina (4) and for E. coli (6). servations were recently reported by Belcour et al. (5). Restriction enzyme analysis. Restriction enzymes were pur- The pl DNA inserted into the prokaryotic plasmid pBR322 chased from Boehringer (Mannheim, Federal Republic ofGer- (6) has been cloned in Escherichia coli, and the hybrid plasmid many). The digestions were performed as recommended by the pSP17 that was isolated from the bacterium was able to induce supplier. Gel electrophoresis was carried out as described (11). senescence injuvenile cultures ofP. anserina (4). Furthermore, Labeling ofDNA. The pl DNA and pBR322 were labeled in heteroduplex analysis and DNA-DNA hybridization (Southern vitro by nick translation (12) with the kit (radioactive component blotting method) have established that pl DNA is an integral [32P]dCTP) from Amersham Buchler (Braunschweig, Federal part of mtDNA from juvenile mycelia of P. anserina (7, 8). Republic of Germany). Specific activities of about 107 cpm/pug The discovery ofpl DNA ofmitochondrial origin in a eukary- of DNA were obtained. otic organism has raised the following questions. (i) Is the pl Blotting and DNA-DNA hybridization. DNA was separated DNA of P. anserina a true plasmid, having the characteristics in agarose gels, transferred to nitrocellulose strips (Sartorius, attributed to bacterial plasmids-i.e., self-replication and G6ttingen, Federal Republic ofGermany) by the Southern (13) expression? The latter has already been shown for pl DNA by procedure, and hybridized to labeled DNA probes as described induction of senescence in juvenile cultures through transfor- (7). The strips were autoradiographed with Kodak X-Omat AR mation experiments (4). Experimental evidence for pl DNA self- film at -70'C for various times. replication will be given in data confirming that pl DNA con- Determination off3-lactamase. The method used was modi- tains an origin of replication (replicon). (ii) Will it be possible fied from those of O'Callaghan and Morris (14) and Hollenberg to use this naturally occurring pl DNA as a vector for the transfer (15). To 3-4 g of mycelium (wet weight), equal amounts of 100 mM phosphate buffer (pH 7.0), designated buffer P1, and glass The publication costs ofthis article were defrayed in part by page charge beads (diameter, 0.1 mm) were added for disruption (30 sec in payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: pl DNA, plasmid-like DNA; kb, kilobase(s). 3641 Downloaded by guest on September 30, 2021 3,542 Microbiology: Stahl et aL Proc. Nati. Acad. Sci. USA 79 (1982) a homogenizer; Braun, Melsungen, Federal Republic of Ger- and (Fig. 1) comprised the following criteria: (i) there were no many). After centrifugation at 10,000 x g for 10 min, the su- homologous sequences of both tester DNAs (pBR322 and pl pernatant (0.1-1.8 ml, variable in (3-lactamase content) was DNA) with nuclear DNA; (ii) as expected, the pl DNA hybrid- made up to 1.9 ml with buffer Pi and mixed with 0. 1 ml ofbuffer ized only with mtDNA from which it originated; and (iii) the Pi containing nitrocephin (5 mg in 10 ml), a chromogenic de- "new" DNA species found after transformation in P. anserina rivative of cephalosporine. (3-Lactamase converts the yellow had homologous sequences with both testers (pBR322 and pl color of this substrate into a red cleavage product that can be DNA)-i.e., the component sequences of pSP17. measured by absorbance at 390 nm. (Nitrocephin was a gift from These results can be considered as strong evidence for the Glaxo Research, Greenford, England.) replication ofthe hybrid plasmid pSP17 in P. anserina because Containment. All transformation experiments were carried the bacterial plasmid pBR322 is not able to amplify in this fun- out under L2/B1 conditions according to the "Richtlinien zum gus. From this it follows that pl DNA must contain the replicon SchutzvorGefahren durch invitro rekombinierte Nukleinsauren" that is able to initiate the replication of the hybrid DNA mol- of the Bundesminister fur Forschung und Technologie. ecule pSP17 in P. anserina. Biological evidence. In order to ascertain whether, after its RESULTS amplification in the eukaryote, the hybrid plasmid pSP17 had In the assessment of the self-replication potential of pl DNA, retained its capacity to replicate and be expressed in a prokary- the cloning ofthe hybrid plasmid pSP17 (consisting ofpBR322 otic system, this plasmid derived from both the wild strain and and two copies of pl DNA) in E. coli is not determinative be- gr/viv was used for transformation ofE. coli. Because the only cause the replication origin of such a hybrid molecule might available marker of pSP17 is the ,B-lactamase gene, ampR, E. stem from the prokaryotic part of this vector. Therefore, we coli cells were screened for ampicillin-resistant transformants. attempted to clone this plasmid in P. anserina, where only the For a further characterization, the DNA of the E. coli clones eukaryotic part may initiate replication. It was established in was isolated and submitted to restriction analysis and Southern early experiments (4) that the prokaryotic part of this vector blotting (Table 1). In addition hybrid plasmid pSP24, which (pBR322) is neither amplified nor expressed when transferred contains only one copy ofpl DNA, was assayed under the same to P. anserina. conditions. During the course of these experiments, the possibility of The data from Table 1 reveal the following facts. (i) As com- expression of the genetic information ofthe prokaryotic part of pared with the control (pBR322), a rather high rate oftransfor- pSP17 in a eukaryotic host could be tested (10). In this context, mation in E. coli was obtained with the hybrid plasmid pSP17. it must be recalled that the plasmid pBR322 carries, in addition This means that the cloning of this plasmid in P. anserina had to the tetracycline-resistance gene whereby the pl DNA is in- not altered its capacity to amplify and to be expressed in the serted, a gene causing resistance to ampicillin and coding for prokaryotic host. (ii) As restriction analysis shows, pSP17 has ,¢lactamase. evidently not been reduced in size because the number ofkilo- These investigations were handicapped by the following fact. bases has remained rather constant (resolution limit, ±200 base Because of the regular occurrence of senescence in the wild pairs). (iii) The data from the Southern blotting technique pre- strain, after infection of protoplasts, many regenerates show a sent a further confirmation, because there is homology between decrease of the life span, involving both transformed and non- both the bacterial plasmid and the pl DNA of P. anserina. (iv) transformed early agers. As reported (4), this difficulty can be The fact that pSP24 gives comparable results shows that evi- overcome by using the double mutant grisealvivax (gr/viv) in dently only one copy of pl DNA is sufficient for the hybrid to which the onset of ageing seems to be delayed indefinitely, as function as a plasmid. it is still alive after more than 3 years ofuninterrupted mycelial growth. Therefore, after protoplast infection, each senescent clone of gr/viv is the result of a transformation. In all experi- pBR322 p1 I)NA ments, both the wild strain and the double mutant gr/viv were pSP mt n pBR pSP nit n1 p1 used as hosts. 17" 322 17: Replication of the Hybrid Plasmid pSP17. In these experi- kb (Control l I(C on tro h ments, protoplasts produced from juvenile mycelia were in- 23.7- fected with the hybrid plasmid pSP17, which was cloned previously in E. coli. Regenerated mycelial clones were tested for senescence, and up to six transformed clones were chosen for 9.5 further analysis from each experiment. 6.7T Physical evidence. In four experiments, the DNA spectrum based on buoyant density profiles was determined by analytical ultracentrifugation. In addition to DNA species normally oc- 4:3 curring in these strains (6), an additional DNA species was found in the wild strain and the double mutant gr/viv. From its buoyant density of 1.704 g/cm3, this additional species clearly rep- resents the hybrid plasmid for which the same value (1.705 g/ cm3) was found in experiments involving E. coli. This confirms that the hybrid plasmid pSP17 is replicated in P. anserina as 2,3- well. 2.0 -- Molecular evidence. In order to support this hypothesis, the was submit- FIG. 1. DNA-DNA hybridization by the Southern technique. After pSP17 isolated by density gradient centrifugation restriction with Sal I, the DNA fragments originating from trans- ted to restriction enzyme analysis. In three experiments it was formed gr/viv strains were separated by electrophoresis and hybrid- found that, according to restriction pattern and additive length ized with bacterial plasmid pBR322 (Left) and with pl DNA of P. an- for fragments, the DNA species was identical with hybrid plas- serina (Right). pSP17, DNA obtained after cloning in P. anserina; mt, mid pSP17. A final proof was obtained from Southern blotting mitochondrial DNA of P. anserina; n, nuclear DNA of P. anserina. Downloaded by guest on September 30, 2021 Microbiology: Stahl et aL Proc. Natl. Acad. Sci. USA 79 (1982) 3643 Table 1. Tranformation of E. coli with hybrid plasmids Table 2. ,B-Lactamase activity in transformants Plasmid DNA from ,&lactamase transformed E. coli activity,* A390 clones Transformed per min/g of Strain with mycelium Transfor- Hybridization mation with P. anserina gr/viv pSP17 0.007 ± 0.001 DNA rate Size, pl Wild type pSP17 0.005 ± 0.002 Donor source x 103* kb pBR322 DNA Wild type pSP24 0.020 ± 0.002 gr/viv - 0 P. anserina gr/viv pSP17 6.6 9.1 + + Wild type - 0 gr/viv mtDNA 0 Saccharomyces or cerevisae AH22 pMP78 0.380 ± 0.002 Wild type pI DNA 0 P. anserina gr/viv pKP402 0.005 ± 0.001 Wild type pSP24 30 6.7 + + E. coli SF8 pBR325 2.530 ± 0.06 E. coli pBR322 2,000 4.3 + - P. anserina gr/viv pKP402 50 6.7 + + ,B-Lactamase activity in transformants ofP. anserina obtained after infection with hybrid plasmids containing a bacterial gene responsible Transformation ofE. coli with different hybrid plasmids recombined for ampicillin resistance. To transformP. anserina, protoplasts (about in vitro and cloned inP. anserina. They consist of pBR322 and p1 DNA 107 cells per ml) of appropriate mycelia were incubated with DNA (pSP17, pSP24) or a p1 DNA homologous region of the mtDNA (5-10 ,ug/ml) in the presence of CaCl2 (10 mM) and polyethylene glycol (pKP402). For their isolation, the mycelia were ground in liquid ni- 4000 (20%) and regenerated on solid complete medium. Nontrans- trogen and lysed with NaDodSO4 (final concentration, 5%). The eluted formed strains of P. anserina and transformants of S. cerevisiae and DNA was purified by hydroxyapatite chromatography and finally by E. coli served as controls. CsCl density centrifugation. The data are the means of four experi- * Mean value of three determinations. ments. For the characterization of E. coli transformants, up to 20 iso- lates were taken from each experiment. In addition to the bacterial plasmid pBR322, which served as a control, nuclear DNA, mtDNA, and pl DNA can be considered as a functional plasmid, comparable p1 DNA of P. anserina were used as further controls. Because, as ex- to plasmids of bacterial or yeast origin. pected, no transformation was achieved with the nuclear DNA of P. Replication and Expression of Hybrid Plasmid pKP402. As anserina, these data are not listed in the table. an extension * Recipient transformation rate of E. coli. ofthese results, the question arises as to whether it is possible to produce an "artificial" vector by cutting out parts of mtDNA homologous to pl DNA and integrating these with In summary, since the prokaryotic part ofthe hybrid plasmid the bacterial vector. can be shown to replicate in a eukaryotic system only when Sal I endonuclease cleaves the mitochondrial DNA ofP. an- joined to pl DNA, it is evident that pl DNA must have an origin serina into five fragments. The adjacent fragments 4 and 1 both of replication. contain a part of pl DNA: the smaller one is Sal I fragment 4, Expression ofthe Hybrid Plasmid pSP17. In addition to rep- which is 5.05 kb long and contains =25% of the pl DNA, lication of hybrid plasmid pSP17 in both a prokaryote and eu- whereas the Sal I fragment 1 is considerably longer (38 kb). karyote, it is ofinterest to know whether this plasmid also is able Because the smaller fragment is easier to handle in cloning ex- to be expressed in both systems. There is no doubt that the periments, it was chosen for further investigations and inte- bacterial part ofthe hybrid plasmid can be expressed in E. coli, grated into the tetracycline gene of pBR322. This hybrid plas- as shown by ampicillin resistance through the production of /3- mid was termed pKP402 (buoyant density, 1.703 g/cm3; 9.37 lactamase, but the expression of the fungal part cannot be as- kb) and can be amplified by cloning in E. coli (7). sayed because there are no specified marker genes known yet In order to find out whether the hybrid plasmid pKP402 is and the senescent phenotype cannot be expected to occur in able to replicate and to be expressed in a eukaryotic system, E. coli. However, with respect to P. anserina, the fungal part protoplasts of the double mutant gr/viv were infected. The of the plasmid is expressed by causing senescence after trans- regenerates showed a rate of senescence of 2.8 X 10-3, which formation in the wild strain and the nonageing double mutant compared favorably to the frequency of pSP17 transformation gr/viv (4). The transformants were tested at regular intervals (2.8 X 10-3). The pKP402 transformants were assayed for DNA up to one-halfyear and were found without exception to contain spectra and production of /-lactamase, and the following facts the hybrid plasmid; none ofthe tested clones spontaneously lost may be seen in the lower sections of Tables 1 and 2. (i) The the plasmid. hybrid plasmid pKP402 was able to replicate in P. anserina In order to test the expression of the bacterial part ofpSP17 because the DNA spectrum showed a DNA species that, ac- in P. anserina, three transformed clones were chosen (from the cording to its buoyant density of 1.703 g/cm3, was equivalent experiments summarized in Table 1) and assayed for intracel- to pKP402. (ii) The mitochondrial part ofthe hybrid plasmid was lular production ofB-lactamase (Table 2). All transformed clones able to be expressed in P. anserina because senescence was from P. anserina produced ,B-lactamase in amounts comparable transformable. In this context, it may be noted that even the to those in a previously described yeast E. coli system (15). It rather small part ofpl DNA (only 25%) was sufficient to induce should be noted that the enzyme production is not influenced senescence. (iii) The bacterial part of the hybrid plasmid by the initial dose of pl DNA because there is no significant pKP402 was also expressed in P. anserina because all senescent difference between the actions of pSP17 and pSP24; the latter clones produced ,B-lactamase in amounts comparable to those contains only one copy of pl DNA. However, it is interesting in the hybrid plasmid pSP17 transformants. that the hybrid plasmid pSP4, in which pl DNA is inverted, was In order to support these findings, the hybrid plasmid found to replicate in P. anserina, but 3-lactamase was not pKP402 was isolated from six transformed P. anserina clones. found. After restriction with Sal I, the fragments were separated and In summary, the eukaryote P. anserina is able to transcribe hybridized with pl DNA and pBR322, respectively (Figs. 2 and and translate a gene of prokaryotic origin, the ampR gene of 3). Because hybridization occurred in all ofthese blots, the pres- pBR322 coupled to pl DNA. Therewith, evidence is given that ence ofpl DNA and pBR322 in the hybrid plasmid pKP402 after Downloaded by guest on September 30, 2021 3644 Microbiology: Stahl et al. Proc. Natd Acad. Sci. USA 79 (1982) expression of such a hybrid plasmid in both a prokaryotic and pBR322 32PIDNA eukaryotic system. DNA CONCLUSION pBR pKP 322 In mt 402 The experimental data clearly show that a distinct part of the mtDNA from the fungus P. anserina is able to replicate and to be expressed autonomously. It can be used as a vector in a eukaryote because bacterial DNA can be transferred, replicated, and eventually expressed in P. anserina when integrated in this mtDNA region. This finding is significant for the following reasons. (i) Eukaryotic cloning is thus extended to mitochondrial genetic information. This view is supportedby the dataofAtchison et al. (16), who transferred mitochondrially coded oligomycin resistance in the S. cerevisiae system. I.4 (ii) It is most likely that other regions ofPodospora mtDNA 4. may be used to construct a vector, ifthey contain a replication origin. It is indeed possible, based on its overall length of 32 Am, that more than the one replication origin is present in Po- dospora mtDNA. This would be expected because, at least in S. cerevisiae, more than one initiation site for DNA replication has been found (17), although so far only one replication origin has been localized in animal mtDNA (length about 5 Ium) (18). (iii) This brings up the question as to whether it is possible in general to use mtDNA replicons to construct "artificial" eukaryotic vectors. In this context, the observation that an origin of replication from mtDNA ofXenopus laevis when integrated in a yeast vector increases transformation rate (17) is ofinterest. Through the use ofmtDNA vectors, it follows that eukaryotes can be more readily adapted for genetic engineering. On the one hand, one is able to dispense with the bacterial plasmid if it is not desired for practical reasons. On the other hand, one may use a prokaryotic-eukaryotic hybrid plasmid as a "shuttle FIG. 2. Autoradiograms of 32P-labeled pBR322 DNA hybridized plasmid" to clone vice versa in both types of systems. with a "Southern blot," containingSal I-digested DNA: hybrid plasmid The data presented in this paper deserve to be discussed in pKP402, obtained after cloning in P. anserina. pBR322 DNA from E. yet another connection-that is, with respect to the senescence coli was used as the positive control, and nuclear (n) and mitochondrial (mt) DNAs from P. anserina were used as the negative control. syndrome in Podospora. (i) The pl DNA homologous part of mtDNA from juvenile mycelia also is able to induce senescence when engineered into a plasmid, and even a small fragment of cloning in P. anserina was evident. the pl DNA sequence is sufficient for this effect. Obviously In summary, the integration of parts of the mtDNA from P. determination ofsenescence by this pl DNA sequence requires anserina into a bacterial plasmid allows the replication and its liberation from the native mtDNA (i.e., its existence as a free plasmid). (ii) Because pl DNA has now proved to be a true plasmid and to be responsible for induction of senescence, previ- RI S ously published data on transformation (4) are now confirmed at the molecular level. :2pIDNA

Sal I pBR322 We thank Prof. P. A. Lemke (Auburn, AL) for reading the manuscript p1 and the members of our staff for their assistance, especially I. Gode- kb hardt, B. Lfickerath, G. Potberg, and U. Rojek. This work was sup- ported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godes- berg, Federal Republic of Germany. 4.35 1. Rizet, G. & Marcou, D. (1954) C. R. Eighth Int. Congr. Bot. Paris, Sec. 10, pp. 121-128. 2. Marcou, D. (1961) Ann. Sci. Nat. Bot. Biot Veg. 2, 653-764. 3. Stahl, U., Lemke, P. A., Tudzynski, P., Kuick, U. & Esser, K. (1978) MoL Gen. Genet. 162, 341-343. 4. Tudzynski, P., Stahl, U. & Esser, K. (1980) Curr. Genet. 2, 2.40 181-184. 5. Belcour, L., Begel, O., Mosse, M. & Vierny, C. (1981) Curr. Genet. 3, 13-21. 6. Stahl, U., Kuck, U., Tudzynski, P. & Esser, K. (1980) MoL Gen. FIG. 3. (Left) R, SalI restriction pattern ofpKP402 DNA, obtained Genet. 178, 639-646. from E. coli, retransformed with hybrid plasmid pKP402, which was 7. Kuck, U., Stahl, U. & Esser, K. (1981) Curr. Genet. 3, 151-156. cloned in P. anserina. (Right) S, corresponding Southern blot hybrid- 8. Esser, K., Tudzynski, P., Stahl, U. & Kuick, U. (1980) Mot Gen. ized with 32P-labeled pBR322 DNA and pl DNA. Genet. 178, 213-216. Downloaded by guest on September 30, 2021 Microbiology: Stahl et al Proc. Nat. Acad. Sci. USA 79 (1982) 3645 9. Esser, K. (1974) Handb. Genet. 1, 531-551. 15. Hollenberg, C. P. (1979) in Plasmids ofMedical, Environmental 10. Bolivar, F., Rodrigez, R. L., Greene, P. J., Betlach, M. C., Hey- and Commercial Importance, eds. Timmis, K. N. & Puhiler, A. neker, H. L., Boyer, H. W., Corso, J. H. & Falkow, S. (1977) (Elsevier/North-Holland, Amsterdam), pp. 481-492. Gene 2, 95-113. 16. Atchison, B. A., Devenish, R. J., Linnane, A. W. & Nagley, P. 11. Kuck, U., Stahl, U., Lhermitte, A. & Esser, K. (1980) Curr. Ge- (1980) Biochem. Biophys. Res. Commun. 96, 580-586. net. 2, 97-101. 17. Goursot, R., De Zamaroczy, M., Baldacci, G. & Bernardi, G. 12. Kelly, R. B., Cozzarelli, N. R., Deutscher, M. P., Lehman, I. R. (1980) Curr. Genet. 1, 173-176. & Kornberg, A. (1970) J. Bid. Chem. 245, 39-45. 18. Gillum, A. M. & Clayton, D. A. (1979)J. Mol. Biol. 135, 353-368. 13. Southern, E. M. (1975) J. Mot Biod 98, 503-517. 19. Zakian, V. A. (1981) Proc. Nati Acad. Sci. USA 78, 3128-3132. 14. O'Callaghan, C. H., Morris, A., Kirby, S. & Shingler, A. H. (1972) Antimicrob. Agents Chemother. 1, 283-288. Downloaded by guest on September 30, 2021