VIROLOGY
183,
586-594
(1991)
Formation of Multimers of Linear Satellite RNAs CLIFFORD D. CARPENTER,*
PAMELA J. CASCONE,t
AND
ANNE E. SIMON*+’
*Department of Biochemistry, tProgram in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Received February 14, 199 1; accepted April 22, 199 1 A 22-base region of turnip crinkle virus satellite-RNA C (sat-RNA C) is involved in the accumulation of monomeric and dimeric forms. Deletions within the region inhibited the accumulation of sat-RNA C monomers. However, normal ratios of dimers to monomers occurred if the 22 bases were replaced by 22 unrelated bases or if the location of this region was altered. Therefore, these specific 22 bases are not involved in the accumulation of sat-RNA C monomers. Examination of the sequences at the junctions of multimers of all three turnip crinkle virus sat-RNAs revealed the deletion of bases corresponding to the 3’ and 5’ ends of monomeric units as well as the addition of nucleotides not present in monomers. Based on these results, we present a model to explain the formation of multimers of linear subviral RNAs associated with turnip crinkle virus. Our model suggests that multimers are formed by the reinitiation of replication by the replicase before release of the nascent strand. We have previously proposed the same mechanism for the formation of defective interfering RNAs, chimeric sat-RNAs, and sat-RNA recombinants in the turnip crinkle virus system (Cascone, Carpenter, Li, and Simon. (1990). f!tdBo 1. 9, 1709-l 715). o 1991 Academic Pws, I~C.
INTRODUCTION
from sat-RNA D (194 bases) by the inclusion of a 20base insert near its 3’ end flanked by 1 g-base repeats of a sequence present once in sat-RNA D. There is no sequence similarity between TCV genomic RNA and sat-RNA D except for the terminal seven nucleotides at the 3’ terminus. Full-length transcripts of sat-RNA C synthesized in vitro are infectious when combined with a helper virus inoculum composed of total RNA isolated from turnip previously infected with TCV genomic RNA and sat-RNA D (Simon and Howell, 1987). SatRNA D is typically included in the helper virus inoculum since it is difficult to keep virus free of this sat-RNA (Li and Simon, 1990). Sat-RNA C containing mutations introduced throughout the middle two-thirds of the molecule is usually infectious, and regions of the sat-RNA affecting virulence, infectivity, and the ratio of monomeric and dimeric forms have been identified (Simon et al., 1988). All three TCV sat-RNAs, as well as TCV DI RNAs, are normally found in infected leaf tissue in both monomeric and multimeric forms (Altenbach and Howell, 1981; Li et al., 1989). Since linear and circular satRNAs in other systems are present as both monomers and oligomers (Kiefer et a/., 1982; Linthorst and Kaper, 1984b; Burgyan and Russo, 1988; Davies eta/., 1990), a rolling circle mechanism of replication has been postulated for small infectious RNAs (Branch and Robet-tson, 1984; lshikawa et a/., 1984; Branch et al., 1988; Davies et al., 1990). In this model, greater than unit length RNAs are generated from a circular, positive, infectious RNA strand. Oligomeric plus strands are synthesized from oligomeric minus strand templates followed by processing and, in the case of natural circular
Plant RNA viruses are frequently associated with linear or circular subviral RNAs known as satellites (satRNAs). Sat-RNAs, which require a helper virus for infectivity, share little or no sequence similarity with their helper virus (Murant and Mayo, 1982). Sat-RNAs thus differ from a second class of subviral RNAs, defective interfering (DI) RNAs, which are completely or nearly completely derived from the helper virus genome. Although many sat-RNAs attenuate symptoms, several examples of sat-RNAs which intensify symptoms have been reported (Kaper and Waterworth, 1977; Altenbath and Howell, 1981). Turnip crinkle virus (TCV) is unusual among plant and animal viruses in supporting the replication of a number of sat- and DI RNAs. Isolate TCV-M is naturally associated with a small family of related, linear satRNAs, one of which (sat-RNA C) intensifies symptoms on all hosts where TCV produces visible symptoms (Li and Simon, 1990). Two other sat-RNA% sat-RNAs F and D, do not alter the symptoms of TCV. The sequence relationships among the sat-RNAs and the single viral genomic RNA have been described (Simon and Howell, 1986). Briefly, sat-RNA C (356 bases) is composed of two domains, a DI-like domain at the 3’ end (166 bases) similar to two regions at the 3’ end of TCV and a 5’ domain (190 bases) similar to the entire sequence of sat-RNA D. Sat-RNA F (230 bases) differs ’ To whom reprint ment of Biochemistry, 1812. 0042-6822191
requests LGRT,
should Amherst,
$3.00
Copynght 0 1991 by Academic Press, Inc. All rights of reproduction I” any form reserved.
be addressed MA 01003,
at the DepartFax: 413.545-
586
MULTIMERIC FORMS OF LINEAR SATELLITE RNAs
molecules circularization of the unit plus strand. However, with the exception of the sat-RNA associated with tobacco ringspot virus (Linthorst and Kaper, 1984a), no circular form of a linear sat-RNA has been found in infected cells (Linthorst and Kaper, 1984b; Simon and Carpenter, unpublished). The absence of circular forms of linear sat-RNAs has led to the suggestion that linear sat-RNA oligomers may be generated by a process which does not require circular molecules (Linthorst and Kaper, 1984b; Collmer and Kaper, 1985). We recently suggested that a replicase driven copy choice mechanism postulated for the generation of DI RNAs and sat-RNA recombinants in the TCV system might also explain the formation of multimers of TCV subviral RNAs (Cascone er al., 1990; Zhang, Cascone and Simon, unpublished). Our model for recombination and DI RNA formation was based on the analysis of junctions between discontinuous RNA molecules; one of three specific replicase-recognition motifs was always found at the right side of the junctions. Two of these motifs are also located at the 5’ ends of the sat-RNAs and TCV genomic RNA. In this paper, we further analyze a 22-base region of sat-RNA C previously determined to be important for the normal ratio of monomeric to multimeric forms of the sat-RNA. Our results indicate that the 22-base region is not specifically involved in a processing reaction and that monomers may not necessarily be derived from multimers via processing in this system. A further indication that rolling circle formed multimers may not be precursors to processed monomers comes from examination of sequences surrounding multimer junctions of all three TCV sat-RNAs. We now propose that multimers of TCV linear subviral RNAs are not formed by a rolling circle mechanism, but are generated in a manner analogous to the formation of recombinant and DI RNAs. MATERIALS
AND METHODS
Plant inoculations, synthesis of infectious transcripts, and RNA analysis
sat-RNA
Wild-type viral isolate TCV-M, containing sat-RNAs D, F, and C, was originally designated TCV-WT. Helper virus inoculum is a preparation of total RNA from uninoculated leaves of infected plants accumulating TCV genomic RNA and sat-RNA D (Simon and Howell, 1987). Synthesis of infectious sat-RNA transcripts in vitro using Escherichia co/i RNA polymerase in a runoff transcription reaction has been described (Simon and Howell, 1987). The buffer and procedure for inoculation were also as previously described (Li et a/., 1989).
587
Two weeks after mechanical inoculation of the initial two true leaves of 2-week-old turnip cv. Just Right seedlings with transcripts and helper virus, RNA was extracted from young, uninoculated leaves approximately 5 cm in length (Simon and Howell, 1986) and analyzed by electrophoresis on 4% polyacrylamide/ 50% urea gels. Northern hybridizations were performed as previously described (Li et a/., 1989). Northern hybridization filters were exposed to X-ray film for various lengths of time to ensure linearity of film grain saturation. Appropriate exposures were scanned with a soft laser scanning densitometer (Biomed Instruments Inc., Fullerton, CA) and data integrated using software provided by the manufacturer. Bal31 deletions
of sat-RNA
C
Stepwise deletions of sat-RNA C were generated in the plasmid pPM2-2-47M+, which contains a fulllength cDNA of sat-RNA C flanked by Smal sites, located downstream from an E. co/i RNA polymerase promoter (Simon and Howell, 1987). pPMZ-2-47M+ was digested with Ncol, ethanol precipitated, and then treated with the slow form of Bal31 (IBI) for various times according to the manufacturer’s suggested conditions. The extent of Ba131 digestion was monitored by Smal restriction analysis of an aliquot of each time point sample. The remainder of the sample was treated with E. co/i DNA polymerase large fragment (Klenow, Bethesda Research Labs) to generate blunt ends and digested with Pstl (which digests pPM2-2-47M+ at a single site 900 bases upstream from the E. co/i RNA polymerase promoter), and the fragment containing leftward deletions was purified and religated into the appropriate fragment of pPM2-2-47M+ to regenerate intact plasmids. Chemical modification sequencing (Simon and Howell, 1986) or dideoxynucleotide chain termination sequencing (Sequenase, United States Biochemical) was used to identify plasmids with deletions of 1 to 21 bases. Manipulation
of the 22-base region of sat-RNA
C
pPMZ-2-47M+ was digested with SnaBl and Ncol, and the purified 22-base fragment treated with Klenow and deoxynucleotides according to manufacturer’s suggested conditions to generate blunt ends by filling in the Ncol site. The “22-base” region (now 26 bases) was ligated into the Dral site of pPM2-2-47M+, resulting in a duplication of the region, or ligated into the Dral site of pPM2-2-47M+ containing a deletion between the SnaBl and Ncol sites (Simon et al., 1988), resulting in the movement of the 22-base region into a new position 10 nucleotides downstream. pPM2-2-47M+ was
588
CARPENTER, CASCONE, AND SIMON
also digested with SnaBl and Ncol, treated with Klenow, and then ligated to a 22-base sequence derived from the polylinker of pUC19 (see legend to Fig. 2). Mutated sat-RNAs accumulating in plants were sequenced using dideoxynucleotide chain termination methods (Carpenter and Simon, 1990) to assess accurate replication of the transcripts. Cloning sat-RNAs using PCR
D and F multimer
junctions
Five micrograms of total RNA isolated from plants containing TCV and either sat-RNA D or sat-RNA F was hybridized to 100 pmol of an oligonucleotide complementary to position 44 through 59 of sat-RNA D in 40 ~1 of 0.4 M NaCI, 10 mM PIPES, pH 6.8, at 80” for 8 min. The sat-RNA D reaction was slow cooled to 44” and ethanol precipitated. Because of oligonucleotide mismatches, the sat-RNA F reaction was cooled to 34” before precipitation. First strand cDNA synthesis was carried out in a 40-~1 reaction consisting of 1 X PCR buffer (20 mM Tris, pH 8.4,50 mM KCI, 2.5 mM MgCI,, 0.1 mg/ml BSA), 0.8 mM of each deoxyribonucleotide, 50 units of ribonuclease inhibitor (U.S. Biochemicals), and 20 units of MMLV reverse transcriptase (BRL). The reaction was first incubated at 25” for 10 min, followed by 42” for 50 min and 95” for 5 min, and then quick cooled on ice. One hundred fifty-eight microliters of 1 X PCR buffer was added along with 10 pmol of the oligonucleotide described above, 10 pmol of oligonucleotide homologous to bases 104-l 18 of sat-RNAs D and F, and 1 unit of Taq polymerase (Cetus). Incubation was for 30 set at 95’, then 30 set at 40’ and 30 set at 72” (sat-RNA D: 25 cycles; sat-RNA F: 30 cycles). Double-stranded cDNA was analyzed by electrophoresis on 4% nondenaturing polyacrylamide gels. For cloning, the cDNA was ethanol precipitated and then treated with T4 polynucleotide kinase (Biolabs) and Klenow according to manufacturers’ suggested conditions. The blunt-ended, phosphorylated fragments thus generated were ligated to the Smal site of pUCl8 after phenol/chloroform extraction and ethanol precipitation. RESULTS Nucleotide sequence is not important in a region of sat-RNA C involved in monomer accumulation in vivo We previously determined that deletion of a 22-base segment in sat-RNA C (position 79-l 00) resulted in the preferential accumulation of sat-RNA dimers rather than monomers in viva. Analysis of sat-RNA C contain-
ing deletions beginning at position 79 and extending toward the 3’ end indicated that deletion of bases in position 79-83 was sufficient to alter the accumulation of monomers and dimers (Simon et al., 1988). In order to further examine the affects of alterations in this region on accumulation of different sat-RNA forms, stepwise deletions of 2 to 21 nucleotides beginning at the Ncol site (position 100) and continuing toward the 5 end were constructed in pPM2-2-47M+, a plasmid containing a full-length cDNA of sat-RNA C downstream from an E. co/i RNA polymerase promoter (Simon et al., 1988). Turnip leaves were mechanically inoculated with transcripts synthesized in vitro together with helper virus. Two weeks postinoculation, RNA was extracted from single leaves and subjected to electrophoresis on 4% denaturing polyacrylamide gels followed by Northern hybridization analysis (Fig. 1). Hybridization intensity levels vary between lanes due to plant-to-plant differences in total sat-RNA accumulation not correlated with the deletions. Approximately 5% of wild-type sat-RNA C accumulated as a dimer in infected plants. Deletions of 3 to 8 bases resulted in the appearance of large amounts of monomeric satRNA C which migrated more slowly than expected. The mobility shift was due to the downstream insertion of poly(U) residues which is described in the companion paper (Carpenter et a/., 1991). Plants infected with sat-RNA containing deletions of 8 to 21 bases accumulated increased amounts of dimeric sat-RNA C, ranging from 22 to 79% of the total amount of sat-RNA. However, no sharp decrease in the amount of monomeric sat-RNA was observed, unlike our previous observation with deletions beginning at position 79 and proceeding toward the Ncol site (Simon et a/., 1988). In order to better understand the role of the sequence between positions 79 and 100 in the generation of monomeric and dimeric sat-RNA forms in vivo, this region was (1) deleted from its original location and reinserted 10 nucleotides downstream; (2) duplicated by insertion of a second copy 10 nucleotides downstream; (3) replaced with 22 unrelated nucleotides in two different orientations (Fig. 2A). Sat-RNA in which the 22-base region had been moved or duplicated accumulated approximately 1 O-fold and 15-fold less, respectively, than wild-type sat-RNA C in plants (Fig. 28, lanes 2 and 3). However, densitometric scanning of several different exposures of the autoradiogram indicated that all dimer:monomer ratios were in the range of wild-type sat-RNA C (~10% dimers). Thus, none of these alterations resulted in a significant increase in the accumulation of dimers with respect to monomers in infected plants, suggesting that the 22-base sequence itself is not involved in a processing reaction.
MULTIMERIC
FORMS
OF
LINEAR
SATELLITE
RNAs
589
Number 5’ 1-234
5 6
of bases deleted
7 8 9101112141516192021
I
5 4 2
183,
586-594
(1991)
Formation of Multimers of Linear Satellite RNAs CLIFFORD D. CARPENTER,*
PAMELA J. CASCONE,t
AND
ANNE E. SIMON*+’
*Department of Biochemistry, tProgram in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Received February 14, 199 1; accepted April 22, 199 1 A 22-base region of turnip crinkle virus satellite-RNA C (sat-RNA C) is involved in the accumulation of monomeric and dimeric forms. Deletions within the region inhibited the accumulation of sat-RNA C monomers. However, normal ratios of dimers to monomers occurred if the 22 bases were replaced by 22 unrelated bases or if the location of this region was altered. Therefore, these specific 22 bases are not involved in the accumulation of sat-RNA C monomers. Examination of the sequences at the junctions of multimers of all three turnip crinkle virus sat-RNAs revealed the deletion of bases corresponding to the 3’ and 5’ ends of monomeric units as well as the addition of nucleotides not present in monomers. Based on these results, we present a model to explain the formation of multimers of linear subviral RNAs associated with turnip crinkle virus. Our model suggests that multimers are formed by the reinitiation of replication by the replicase before release of the nascent strand. We have previously proposed the same mechanism for the formation of defective interfering RNAs, chimeric sat-RNAs, and sat-RNA recombinants in the turnip crinkle virus system (Cascone, Carpenter, Li, and Simon. (1990). f!tdBo 1. 9, 1709-l 715). o 1991 Academic Pws, I~C.
INTRODUCTION
from sat-RNA D (194 bases) by the inclusion of a 20base insert near its 3’ end flanked by 1 g-base repeats of a sequence present once in sat-RNA D. There is no sequence similarity between TCV genomic RNA and sat-RNA D except for the terminal seven nucleotides at the 3’ terminus. Full-length transcripts of sat-RNA C synthesized in vitro are infectious when combined with a helper virus inoculum composed of total RNA isolated from turnip previously infected with TCV genomic RNA and sat-RNA D (Simon and Howell, 1987). SatRNA D is typically included in the helper virus inoculum since it is difficult to keep virus free of this sat-RNA (Li and Simon, 1990). Sat-RNA C containing mutations introduced throughout the middle two-thirds of the molecule is usually infectious, and regions of the sat-RNA affecting virulence, infectivity, and the ratio of monomeric and dimeric forms have been identified (Simon et al., 1988). All three TCV sat-RNAs, as well as TCV DI RNAs, are normally found in infected leaf tissue in both monomeric and multimeric forms (Altenbach and Howell, 1981; Li et al., 1989). Since linear and circular satRNAs in other systems are present as both monomers and oligomers (Kiefer et a/., 1982; Linthorst and Kaper, 1984b; Burgyan and Russo, 1988; Davies eta/., 1990), a rolling circle mechanism of replication has been postulated for small infectious RNAs (Branch and Robet-tson, 1984; lshikawa et a/., 1984; Branch et al., 1988; Davies et al., 1990). In this model, greater than unit length RNAs are generated from a circular, positive, infectious RNA strand. Oligomeric plus strands are synthesized from oligomeric minus strand templates followed by processing and, in the case of natural circular
Plant RNA viruses are frequently associated with linear or circular subviral RNAs known as satellites (satRNAs). Sat-RNAs, which require a helper virus for infectivity, share little or no sequence similarity with their helper virus (Murant and Mayo, 1982). Sat-RNAs thus differ from a second class of subviral RNAs, defective interfering (DI) RNAs, which are completely or nearly completely derived from the helper virus genome. Although many sat-RNAs attenuate symptoms, several examples of sat-RNAs which intensify symptoms have been reported (Kaper and Waterworth, 1977; Altenbath and Howell, 1981). Turnip crinkle virus (TCV) is unusual among plant and animal viruses in supporting the replication of a number of sat- and DI RNAs. Isolate TCV-M is naturally associated with a small family of related, linear satRNAs, one of which (sat-RNA C) intensifies symptoms on all hosts where TCV produces visible symptoms (Li and Simon, 1990). Two other sat-RNA% sat-RNAs F and D, do not alter the symptoms of TCV. The sequence relationships among the sat-RNAs and the single viral genomic RNA have been described (Simon and Howell, 1986). Briefly, sat-RNA C (356 bases) is composed of two domains, a DI-like domain at the 3’ end (166 bases) similar to two regions at the 3’ end of TCV and a 5’ domain (190 bases) similar to the entire sequence of sat-RNA D. Sat-RNA F (230 bases) differs ’ To whom reprint ment of Biochemistry, 1812. 0042-6822191
requests LGRT,
should Amherst,
$3.00
Copynght 0 1991 by Academic Press, Inc. All rights of reproduction I” any form reserved.
be addressed MA 01003,
at the DepartFax: 413.545-
586
MULTIMERIC FORMS OF LINEAR SATELLITE RNAs
molecules circularization of the unit plus strand. However, with the exception of the sat-RNA associated with tobacco ringspot virus (Linthorst and Kaper, 1984a), no circular form of a linear sat-RNA has been found in infected cells (Linthorst and Kaper, 1984b; Simon and Carpenter, unpublished). The absence of circular forms of linear sat-RNAs has led to the suggestion that linear sat-RNA oligomers may be generated by a process which does not require circular molecules (Linthorst and Kaper, 1984b; Collmer and Kaper, 1985). We recently suggested that a replicase driven copy choice mechanism postulated for the generation of DI RNAs and sat-RNA recombinants in the TCV system might also explain the formation of multimers of TCV subviral RNAs (Cascone er al., 1990; Zhang, Cascone and Simon, unpublished). Our model for recombination and DI RNA formation was based on the analysis of junctions between discontinuous RNA molecules; one of three specific replicase-recognition motifs was always found at the right side of the junctions. Two of these motifs are also located at the 5’ ends of the sat-RNAs and TCV genomic RNA. In this paper, we further analyze a 22-base region of sat-RNA C previously determined to be important for the normal ratio of monomeric to multimeric forms of the sat-RNA. Our results indicate that the 22-base region is not specifically involved in a processing reaction and that monomers may not necessarily be derived from multimers via processing in this system. A further indication that rolling circle formed multimers may not be precursors to processed monomers comes from examination of sequences surrounding multimer junctions of all three TCV sat-RNAs. We now propose that multimers of TCV linear subviral RNAs are not formed by a rolling circle mechanism, but are generated in a manner analogous to the formation of recombinant and DI RNAs. MATERIALS
AND METHODS
Plant inoculations, synthesis of infectious transcripts, and RNA analysis
sat-RNA
Wild-type viral isolate TCV-M, containing sat-RNAs D, F, and C, was originally designated TCV-WT. Helper virus inoculum is a preparation of total RNA from uninoculated leaves of infected plants accumulating TCV genomic RNA and sat-RNA D (Simon and Howell, 1987). Synthesis of infectious sat-RNA transcripts in vitro using Escherichia co/i RNA polymerase in a runoff transcription reaction has been described (Simon and Howell, 1987). The buffer and procedure for inoculation were also as previously described (Li et a/., 1989).
587
Two weeks after mechanical inoculation of the initial two true leaves of 2-week-old turnip cv. Just Right seedlings with transcripts and helper virus, RNA was extracted from young, uninoculated leaves approximately 5 cm in length (Simon and Howell, 1986) and analyzed by electrophoresis on 4% polyacrylamide/ 50% urea gels. Northern hybridizations were performed as previously described (Li et a/., 1989). Northern hybridization filters were exposed to X-ray film for various lengths of time to ensure linearity of film grain saturation. Appropriate exposures were scanned with a soft laser scanning densitometer (Biomed Instruments Inc., Fullerton, CA) and data integrated using software provided by the manufacturer. Bal31 deletions
of sat-RNA
C
Stepwise deletions of sat-RNA C were generated in the plasmid pPM2-2-47M+, which contains a fulllength cDNA of sat-RNA C flanked by Smal sites, located downstream from an E. co/i RNA polymerase promoter (Simon and Howell, 1987). pPMZ-2-47M+ was digested with Ncol, ethanol precipitated, and then treated with the slow form of Bal31 (IBI) for various times according to the manufacturer’s suggested conditions. The extent of Ba131 digestion was monitored by Smal restriction analysis of an aliquot of each time point sample. The remainder of the sample was treated with E. co/i DNA polymerase large fragment (Klenow, Bethesda Research Labs) to generate blunt ends and digested with Pstl (which digests pPM2-2-47M+ at a single site 900 bases upstream from the E. co/i RNA polymerase promoter), and the fragment containing leftward deletions was purified and religated into the appropriate fragment of pPM2-2-47M+ to regenerate intact plasmids. Chemical modification sequencing (Simon and Howell, 1986) or dideoxynucleotide chain termination sequencing (Sequenase, United States Biochemical) was used to identify plasmids with deletions of 1 to 21 bases. Manipulation
of the 22-base region of sat-RNA
C
pPMZ-2-47M+ was digested with SnaBl and Ncol, and the purified 22-base fragment treated with Klenow and deoxynucleotides according to manufacturer’s suggested conditions to generate blunt ends by filling in the Ncol site. The “22-base” region (now 26 bases) was ligated into the Dral site of pPM2-2-47M+, resulting in a duplication of the region, or ligated into the Dral site of pPM2-2-47M+ containing a deletion between the SnaBl and Ncol sites (Simon et al., 1988), resulting in the movement of the 22-base region into a new position 10 nucleotides downstream. pPM2-2-47M+ was
588
CARPENTER, CASCONE, AND SIMON
also digested with SnaBl and Ncol, treated with Klenow, and then ligated to a 22-base sequence derived from the polylinker of pUC19 (see legend to Fig. 2). Mutated sat-RNAs accumulating in plants were sequenced using dideoxynucleotide chain termination methods (Carpenter and Simon, 1990) to assess accurate replication of the transcripts. Cloning sat-RNAs using PCR
D and F multimer
junctions
Five micrograms of total RNA isolated from plants containing TCV and either sat-RNA D or sat-RNA F was hybridized to 100 pmol of an oligonucleotide complementary to position 44 through 59 of sat-RNA D in 40 ~1 of 0.4 M NaCI, 10 mM PIPES, pH 6.8, at 80” for 8 min. The sat-RNA D reaction was slow cooled to 44” and ethanol precipitated. Because of oligonucleotide mismatches, the sat-RNA F reaction was cooled to 34” before precipitation. First strand cDNA synthesis was carried out in a 40-~1 reaction consisting of 1 X PCR buffer (20 mM Tris, pH 8.4,50 mM KCI, 2.5 mM MgCI,, 0.1 mg/ml BSA), 0.8 mM of each deoxyribonucleotide, 50 units of ribonuclease inhibitor (U.S. Biochemicals), and 20 units of MMLV reverse transcriptase (BRL). The reaction was first incubated at 25” for 10 min, followed by 42” for 50 min and 95” for 5 min, and then quick cooled on ice. One hundred fifty-eight microliters of 1 X PCR buffer was added along with 10 pmol of the oligonucleotide described above, 10 pmol of oligonucleotide homologous to bases 104-l 18 of sat-RNAs D and F, and 1 unit of Taq polymerase (Cetus). Incubation was for 30 set at 95’, then 30 set at 40’ and 30 set at 72” (sat-RNA D: 25 cycles; sat-RNA F: 30 cycles). Double-stranded cDNA was analyzed by electrophoresis on 4% nondenaturing polyacrylamide gels. For cloning, the cDNA was ethanol precipitated and then treated with T4 polynucleotide kinase (Biolabs) and Klenow according to manufacturers’ suggested conditions. The blunt-ended, phosphorylated fragments thus generated were ligated to the Smal site of pUCl8 after phenol/chloroform extraction and ethanol precipitation. RESULTS Nucleotide sequence is not important in a region of sat-RNA C involved in monomer accumulation in vivo We previously determined that deletion of a 22-base segment in sat-RNA C (position 79-l 00) resulted in the preferential accumulation of sat-RNA dimers rather than monomers in viva. Analysis of sat-RNA C contain-
ing deletions beginning at position 79 and extending toward the 3’ end indicated that deletion of bases in position 79-83 was sufficient to alter the accumulation of monomers and dimers (Simon et al., 1988). In order to further examine the affects of alterations in this region on accumulation of different sat-RNA forms, stepwise deletions of 2 to 21 nucleotides beginning at the Ncol site (position 100) and continuing toward the 5 end were constructed in pPM2-2-47M+, a plasmid containing a full-length cDNA of sat-RNA C downstream from an E. co/i RNA polymerase promoter (Simon et al., 1988). Turnip leaves were mechanically inoculated with transcripts synthesized in vitro together with helper virus. Two weeks postinoculation, RNA was extracted from single leaves and subjected to electrophoresis on 4% denaturing polyacrylamide gels followed by Northern hybridization analysis (Fig. 1). Hybridization intensity levels vary between lanes due to plant-to-plant differences in total sat-RNA accumulation not correlated with the deletions. Approximately 5% of wild-type sat-RNA C accumulated as a dimer in infected plants. Deletions of 3 to 8 bases resulted in the appearance of large amounts of monomeric satRNA C which migrated more slowly than expected. The mobility shift was due to the downstream insertion of poly(U) residues which is described in the companion paper (Carpenter et a/., 1991). Plants infected with sat-RNA containing deletions of 8 to 21 bases accumulated increased amounts of dimeric sat-RNA C, ranging from 22 to 79% of the total amount of sat-RNA. However, no sharp decrease in the amount of monomeric sat-RNA was observed, unlike our previous observation with deletions beginning at position 79 and proceeding toward the Ncol site (Simon et a/., 1988). In order to better understand the role of the sequence between positions 79 and 100 in the generation of monomeric and dimeric sat-RNA forms in vivo, this region was (1) deleted from its original location and reinserted 10 nucleotides downstream; (2) duplicated by insertion of a second copy 10 nucleotides downstream; (3) replaced with 22 unrelated nucleotides in two different orientations (Fig. 2A). Sat-RNA in which the 22-base region had been moved or duplicated accumulated approximately 1 O-fold and 15-fold less, respectively, than wild-type sat-RNA C in plants (Fig. 28, lanes 2 and 3). However, densitometric scanning of several different exposures of the autoradiogram indicated that all dimer:monomer ratios were in the range of wild-type sat-RNA C (~10% dimers). Thus, none of these alterations resulted in a significant increase in the accumulation of dimers with respect to monomers in infected plants, suggesting that the 22-base sequence itself is not involved in a processing reaction.
MULTIMERIC
FORMS
OF
LINEAR
SATELLITE
RNAs
589
Number 5’ 1-234
5 6
of bases deleted
7 8 9101112141516192021
I
5 4 2
