Reverse transcription of yeast double-stranded RNA and ribosomal RNA using synthetic oligonucleotide primers

Biochimica et Biophysica A cta, 739 (1983) 122-131

122

Elsevier Biomedical Press BBA 91165

REVERSE T R A N S C R I P T I O N OF YEAST D O U B L E - S T R A N D E D RNA AND R I B O S O M A L RNA USING SYNTHETIC OLIGONUCLEOTIDE PRIMERS BILLY L. B R I Z Z A R D

*

and SIWO

R.

DE K L O E T **

Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306 (U.S.A.) (Received June 24th, 1982)

Key words: Reverse transcription," double-stranded RNA; rRNA; Oligonucleotide primer; (Yeast)

The ability of the four oligodeoxyribonucleotide primers oligo(dT)lz_ ts, oligo(dA)t2-18, oligo(dG)n2-18 and oligo(dC)n2_18 to act as primers for avian myeloblastosis virus reverse transcriptase on denatured yeast double-stranded (ds) RNA templates was investigated. Oiigo(dT) and oligo(dA) were found to prime the synthesis of 1.1 and 1.0 kb reverse transcripts, respectively, using denatured M dsRNA as a template. The oligo(dT)- and oligo(dA)-primed cDNAs of M dsRNA hybridized to the region of the M dsRNA that encoded the killer toxin and to each other. Addition of oligo(dT) to reverse transcription reactions of denatured L dsRNA produced a 4.3 kb cDNA. During the course of this investigation oligo(dC) was observed to he a highly efficient primer for reverse transcription of yeast 18 S ribosomal RNA. Oligo(dC) primed the synthesis of a 1.0 kb transcript of 18 S rRNA which hybridized to the large EcoRl fragment of the 18 S rRNA gene. Reverse transcription of double-stranded RNA and 25 S ribosomal RNA was found to occur to some extent in the absence of added oligonueleotide primer.

Introduction Killer strains of Saccharomyces cerevisiae secrete a toxin which kills sensitive strains [ 1,2]. The toxin and immunity are encoded by a 1830 bp doublestranded (ds) RNA plasmid designated M [2,3]. The M dsRNA molecule has been reported to contain a 190 bp region which is essentially A + U [4]. This region of A + U subdivides the molecule into two regions of 1000 and 630 bp [5]. The 1000 bp region has been shown to encode the killer toxin [5]. Most yeast strains, killer or nonkiller,

* Present address: Department of T u m o r Virology, M.D. Anderson Hospital and T u m o r Institute, Houston, TX 77030, U.S.A. ** To whom correspondence should be addressed. Abbreviations: AMV, avian myeloblastosis virus; SSC, standard saline citrate solution (0.15 M NaCI/0.015 M trisodium citrate, concentrations given as multiples of this). 0167-4781/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press

contain another dsRNA plasmid 4.8 kbp in length designated L [2]. The L plasmid encodes the viral coat protein [6]. The L plasmid is not known to contain any extended region of high A + U content. The synthesis of a double-stranded cDNA copy of the M dsRNA will make it possible to clone the dsRNA-encoded killer toxin as doublestranded cDNA. AMV reverse transcriptase is an RNA-directed DNA polymerase which requires a primer for activity [7-9]. Oligo(dT) is often used as a primer for reverse transcription of eukaryotic messenger RNAs which possess a 3'-poly(A) tail [10], However, other types of RNA lack poly(A) tails. Reverse transcription of nonmessenger RNAs has been accomplished by the enzymatic addition of a poly(A) tail [11,12], or by the use of synthetic or natural primers complementary to sequences within the RNA [13,14]. We have investigated the ability of the four

123 oligodeoxyribonucleotide primers oligo(dT)12_ls, oligo(dA)12_ls, oligo(dG)12_t8 and oligo(dC)12_18 to act as primers for reverse transcription of denatured dsRNA. Oligo(dT) and oligo(dA) were found to be effective primers for reverse transcription of denatured M clsRNA. The oligo(dT)- and oligo(dA)-primed cDNAs were shown to hybridize to the 1000 bp region of the M dsRNA which encode to the killer toxin and to each other. In the course of this study we observed that oligo(dC) is an extremely effective primer for reverse transcription of yeast 18 S ribosomal RNA. The 1.0 kb oligo(dC)-primed cDNA hybridized to the large EcoRI restriction fragment of the yeast 18 S rRNA gene which contains the 5'-terminus of the mature 18 S rRNA [15]. Materials and Methods

Cultivation of yeast. The yeast strain used in this study was the superkiller, T158C, kindly provided by Dr. Gerald Fink. Yeast cells were grown in complete medium as previously described [16]. Nucleic acid isolation. Double-stranded RNA was extracted by the method of Fried and Fink [4] and purified by CF-I 1 cellulose chromatography using the modification of Franklin [17]. In order to separate L and M dsRNAs, aliquots of column purified total dsRNA were subjected to electrophoresis on 2% agarose gels in buffer E (40 mM Tris-CH3COOH, pH 7.2/2 mM EDTA). L and M dsRNAs were extracted from the gel by the freeze and squeeze method [18] followed by phenol extraction and ethanol precipitation. Purified L and M dsRNAs were dissolved and stored in 2 mM EDTA. Prior to reverse transcription, dsRNA was denatured by heating to 100°C for 3 min in 2 mM EDTA, pH 7.0, and quickly chilled on ice [3]. For the preparation of mRNA, rRNA and DNA, mid-log phase cells were harvested by centrifugation and converted to protoplasts using snail enzyme (fl-glucuronidase, Calbiochem). The protoplasts were lysed with 0.5% SDS in a buffer containing 100 mM NaCI, 50 mM Tris-HCl (pH 8.8) and 2 mM EDTA and extracted with phenol/chloroform. The aqueous phase was collected and precipitated with ethanol. The pellet was redissolved in SSC and an equal volume of 4 M LiCI/2 × SSC was added to precipitate high-

molecular-weight single-stranded RNA. Following centrifugation, the pellet was redissolved in 0.4 M N a C I / 4 mM E D T A / 1 0 mM Tris-HC1 (pH 7.9)/0.2% SDS and fractionated into polyadenylate-containing, poly(A) ÷, and nonpolyadenylatecontaining, poly(A) , RNA by chromatography on poly(U)-Sepharose (Pharmacia). Aliquots of poly(A)- RNA were subjected to sucrose gradient centrifugation on a 15-30% ( w / v ) gradient in a buffer containing 100 mM NaC1/20 mM sodium acetate (pH 6.0)/2 mM EDTA. The fractions containing 18 and 25 S rRNA were recovered by ethanol precipitation. DNA was recovered from the 2 M LiCI supernatant by ethanol precipitation of the nucleic acid. The pellet was redissolved in 0.2 × SSC and incubated with 50 /~g/ml pancreatic RNAase (Sigma) at 37°C for 30 min. The solution was then heated to 60°C and 100 /~g/ml pronase (Calbiochem) and SDS to 0.1% were added. After 1 h of incubation the solution was extracted with phenol/chloroform. The aqueous phase was collected and dialyzed against 2 mM EDTA, pH 7.0. CsC1 was added to a final density of 1.7 g / c m 3 in a buffer containing 20 mM Tris-HC1 (pH 7.2), 2 mM EDTA and 10/~g/ml ethidium bromide. The gradients were centrifuged for 72 h at 36000 rev./min in an A-321 rotor in an International Ultracentrifuge. Fractions containing high-molecular-weight DNA were located by their ethidium bromide fluorescence, pooled and mixed with an equal volume of isopropanol to remove ethidium bromide. The sample was then dialyzed against 2 mM EDTA, pH 7.0. The dialysate was precipitated with ethanol, and the pellet was repeatedly washed with 70% ethanol before being redissolved. cDNA synthesis. AMV reverse transcriptase was obtained from Dr. Joseph Beard (Life Sciences, Inc., St. Petersburg, FL) and from Bethesda Research Laboratories (BRL). The reaction conditions were as described by Schibler et al. [19] for full-length synthesis of cDNA. Typically, reactions were performed in a final volume of 50 /~1 and contained 800 ~M each of dTTP, dATP, dGTP, 200 ~M dCTP, 10/~Ci [tx-32p]dCTP (3000 C i / m mol), 75 units of BRL reverse transcriptase or 75 units of Life Sciences reverse transcriptase, 1 /tg RNA, 1 /~g oligonucleotide primer (Miles), 50 ~ g / m l actinomycin D, 50 mM Tris-HC1 (pH 8.3),

124

10 mM dithiothreitol, 10 mM magnesium acetate, 50 mM NaC1 and 100 t t g / m l bovine serum albumin. Samples were incubated at 42°C for 40 min and terminated by the addition of EDTA to 20 mM. The samples were phenol extracted as described by Schibler et al. [19]. The aqueous phase was then ethanol-precipitated following the addition of 20 #g Escherichia coli carrier tRNA and redissolved in 30 mM N a O H for alkaline agarose gel electrophoresis. Samples to be used as hybridization probes were redissolved in 0.3 M N a O H and incubated at room temperature for 1 h. Following neutralization with 10 M acetic acid the samples were purified by Sephadex G-100 chromatography.

Restriction endonuclease digestions and 5'-end labeling with polynucleotide kinase. The hybrid plasmid YR p7 which contains the 1.4 kb yeast trpI gene and pBR 322 was the generous gift of Dr. R.W. Davis. The plasmid was purified from E. coli C600 essentially as described by Colman et al. [20]. EcoRI and HindIII restriction enzymes were obtained from BRL. Digestions were performed as described in the BRL catalog. For molecular weight markers 2 /~g of plasmid YR p7 and 5 /~g of )~-DNA (Miles) were digested with EcoRI and HindIII, respectively. Restricted DNA was 5'-end labeled by exchange using polynucleotide kinase (BRL) as described by Maxam and Gilbert [21]. EcoRI digestion of yeast genomic DNA was performed in the same manner and the samples were subjected to electrophoresis on 1% agarose gels. Gel electrophoresis. Agarose gel electrophoresis was performed in buffer E. The gels were stained with 0.5 ~ g / m l ethidium bromide and photographed under ultraviolet illumination using a Polaroid MP-3 camera. Alkaline agarose gel electrophoresis was performed as described by McDonnel et al. [22] using 30 mM N a O H / 2 mM EDTA in the tray buffer and the gel. Radioactive cDNAs were visualized by autoradiography at - 7 0 ° C using Kodak XR-5 film and a Dupont Lighting-Plus intensifying screen. Partial Sj nuclease digestion of M dsRNA. M dsRNA (5~g) purified as described above was digested with S~ nuclease (BRL) under partially

denaturing conditions (65°C) as described by Welsh and Leibowitz [5]. The S t buffer contained 20 mM NaC1, 40 mM sodium acetate (pH 4.5) and 10 mM ZnSO 4. S I nuclease was added at a ratio of 3 units per ~g of M dsRNA. Digested RNA was then subjected to electrophoresis on a 2% agarose gel in buffer E. Gel transfer hybridization. Eco RI-digested yeast genomic DNA (5 ~g) was subjected to electrophoresis on a 1% agarose slab gel and transferred to nitrocellulose as described by Southern [23]. Reverse transcripts of 18 and 25 S rRNA were hybridized separately to filters in the hybridization buffer described by Wahl et al. [24] which contained 50% formamide, 5 x SSC, 10 mM sodium phosphate (pH 6.5), 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% poly(vinyl pyrrolidone), 0.1% SDS and 200 ~ g / m l sonicated-denatured salmon sperm DNA at 42°C. Sl-digested M dsRNA was subjected to electrophoresis on a 2% agarose tube gel and transferred to DBM-paper as described by Alwine et al. [25]. Oligo(dT)- and oligo(dA)-primed cDNAs of denatured M dsRNA were hybridized separately to immobilized RNA in the same buffer described above at 42°C. After hybridization the filters were washed in 2 × SSC/0.1% SDS and autoradiography was performed as described above. Solution hybridizations. Solution hybridization of the oligo(dT)- and oligo(dA)-primed cDNAs of denatured M dsRNA was performed by a modification of the method of Weaver and Weissmann [26]. Oligo(dT)- and oligo(dA)-primed reverse transcripts of denatured M dsRNA were purified as described above and collected by ethanol precipitation. The transcripts were redissolved in 99% formamide and an equal volume of buffer, containing 40 mM sodium acetate (pH 6.4), 1.5 M NaC1, 200 ~ g / m l sonicated-denatured salmon sperm DNA and 1 mM EDTA, was added. Hybridization was performed at 42°C. Aliquots of the hybridized samples were diluted 20-fold in S I buffer [5] and digested for 1 h at 37°C with 1000 units/ml S l nuclease. The samples were precipitated with 5% trichloroacetic acid/10 mM sodium pyrophosphate and collected on Whatman 3MM filters. The filters were counted in 0.4% omnifluor in toluene in a Beckman liquid scintillation counter.

125

Results D e n a t u r e d d s R N A was transcribed with A M V reverse transcriptase o b t a i n e d from Life Sciences a n d BRL (Table I). T r a n s c r i p t i o n was performed as described by Schibler et al. [19] for full-length t r a n s c r i p t i o n of i m m u n o g l o b u l i n messenger R N A . M a x i m u m i n c o r p o r a t i o n was observed after inc u b a t i o n at 4 2 ° C for 40 min. For L d s R N A , oligo(dG) was the most effective primer when the Life Sciences enzyme was used. Interestingly, this result is n o longer seen when the BRL enzyme is used (Table I).

Reverse transcription of ribosomal RNA Reverse t r a n s c r i p t i o n of p o l y ( A ) - e n r i c h e d single-stranded R N A (Table I) showed the expected primer activity of oligo(dT) (55 ng c D N A synthesized, 5.5% yield) and, in addition, the surprising result that oligo(dC) was almost as effective (38 ng c D N A synthesized, 3.8% yield for the BRL enzyme). Oligo(dC) (but not oligo(dT)) p r i m e d reverse transcription of n o n - p o l y a d e n y -

lated single-stranded (ss) R N A which suggested that oligo(dC) was not p r i m i n g the reverse transcription of messenger R N A . In order to test this hypothesis ribosomal R N A was made free of poly(A) R N A by chromatograp h y on poly(U)-Sepharose. 18 a n d 25 S r R N A were then purified by sucrose density gradient centrifugation a n d used as templates with each of the four primers. Oligo(dC) was by far the most effective primer for both 18 (9.5% yield) a n d 25 S (5.7% yield) r R N A (Table I). This result suggests that oligo(dC) primes the reverse transcription of yeast ribosomal R N A . Reverse transcription of ribosomal R N A has b e e n reported to occur in the absence of exogen o u s primer [27]. This was f o u n d to be the case for 25 S r R N A , b u t not for 18 S r R N A (see below). In order to characterize the products of reverse t r a n s c r i p t i o n the molecular size of the transcripts was d e t e r m i n e d by alkaline agarose gel electrophoresis. T h e p r o d u c t of reverse transcription reactions of 18 a n d 25 S r R N A to which oligo(dC)

TABLE I REVERSE TRANSCRIPTION OF YEAST RNA Reverse transcription of the indicated RNA samples was performed as described in Materials and Methods using 75 units of AMV reverse transcriptase (the results using the Life Sciences enzyme are the upper values and the results using the BRL enzyme are the lower values). Aliquots were taken at 0 and 40 rain of incubation and precipitated with trichloroacetic acid and counted as described. The values shown represent the total cpm [a-a2p]dCTP incorporated after subtraction of the zero-time value. A dash indicates the reaction was not performed. Sample

oligo(dT)

oligo(dA)

oligo(dG)

Den. L

59 280 15560

48 550 16390

116960 16870

78 800 20 230

15 300

Den. M

18550 22850

26150 23 800

18900 7 100

20320 24120

26000

Poly(A)+

290670 56 740

33 000 -

64 000 -

343 860 39170

-

Poly(A)-

43 350

47 370

53 750

267 040

-

25 S rRNA

60 850 -

50 336 -

31 300 -

287 870 59580

21 580

18 S rRNA

20000 -

22940 -

16410 -

352000 98310

8040

No RNA

. 5 040

.

. 13 850

. 1630

oligo(dC)

No primer

. 5 440

-

126

A

B T

A

~

C

hLR

C

B

T C

N,F~ C

18S

25S

43-

i !iiiiiiiii iii - h4

kb

-'~3

kb

kb 1.4-

kbp

O ,

2

,

4

,

,

7

.

,

':

--I.4

t

7'

Fig. 1. A. Alkaline agarose gel electrophoresis of labeled cDNAs (BRL enzyme). Lanes 1-4 oligo(dT), oligo(dA), oligo(dG) and oligo(dC) without template. Lane 5:25 S rRNA without primer. Lane 6:25 S rRNA with oligo(dC). Lane 7:18 S rRNA without primer. Lane 8:18 S rRNA with oligo(dC). Lane 9: 32p-labeled 1.4 kb E c o R l fragment of YR p7. B. Alkaline agarose gel electrophoresis of oligo(dT)-primed reverse transcription of poly(A) ÷ RNA (lane 1) and oligo(dC)-primed reverse transcription of poly(A) + RNA (lane 2), The BRL enzyme was used. The markers are labeled EcoRl fragments of YR p7.

was added was a 1.0 kb cDNA in each case (Fig. 1A). However, for 25 S rRNA the 1.0 kb cDNA was also synthesized in the absence of added primer. For 18 S rRNA no high molecular weight cDNA is synthesized in the absence of primer. Therefore, reverse transcription of yeast 25 S rRNA is not primer specific. As expected, oligo(dT)-primed reverse transcription of poly(A)-enriched ssRNA produced a heterogeneous population of cDNAs (Fig. 1B). In contrast, oligo(dC)-primed reverse transcription of poly(A)-enriched ssRNA produced a cDNA of distinct size (1.0 kb). Some incorporation of label in the absence of template was seen with each of the four primers (Table I). However, no high molecular weight c D N A is synthesized (Fig. IA). Hybridization of the reverse transcripts of ribosomal RNA to EcoRI fragments of the ribosomal RNA gene cluster In an attempt to localize the site of initiation of reverse transcription of 25 S rRNA and the prim-

2

5

C Fig. 2. A. Agarose gel electrophoresis of 5 ~tg of EcoRI-digested yeast genomic DNA stained with ethidium bromide (lane 1). B. Southern hybridization of E c o R l digested DNA. Lane 2: The oligo(dC)-primed cDNA of 18 S rRNA was hybridized following digestion with alkali and G-100 chromatography. Lane 3: cDNA of 25 S rRNA (with added oligo(dC)) hybridized following alkali digestion and G-100 chromatography. The BRL enzyme was used. The approximate specific activity of the probes was 106 cpm//~g. C. E c o R l restriction map of the yeast ribosomal RNA gene cluster [15]. The approximate locations of the 5'- and 3'-ends of 25 and 18 S rRNA are indicated.

ing site of oligo(dC) on 18 S rRNA, the reverse transcript of 25 S rRNA and the oligo(dC)-primed reverse transcript of 18 S rRNA were hybridized with EcoRI-digested yeast genomic DNA. EcoRI digestion of the yeast ribosomal RNA gene cluster produces seven restriction fragments [15,28,29]. The EcoRI restriction map of the yeast ribosomal gene cluster is shown in Fig. 2C. EcoRI digestion of DNA from strain T158C is shown in Fig. 2A. Fragments A, F and E originate from the 25 S rRNA gene. Fragments C and D originate from the 18 S rRNA gene. * * Fragments F and G were visible in the gel but are not visible in the photograph in Fig. 2A.

127

Following electrophoresis EcoRI-digested D N A was transferred to nitrocellulose and hybridized separately with the oligo(dC)-primed reverse transcript of 18 S r R N A and the reverse transcript of 25 S r R N A (oligo(dC) was added to the reaction in an attempt to increase the yield of cDNA). The oligo(dC)-primed transcript of 18 S r R N A hybridized predominantly to EcoRI fragment C (Fig. 2B). Some hybridization to fragment A is seen, however, indicating contamination with 25 S rRNA. Likewise, the reverse transcript of 25 S r R N A hybridized predominantly to fragment A. The strong hybridization of the oligo(dC)-primed reverse transcript of 18 S rRNA, taken together with the greater incorporation seen with this template (Table I), suggests that oligo(dC) is a highly efficient primer for reverse transcription of yeast 18 S rRNA.

Reverse transcription of dsRNA All four primers were active when denatured L d s R N A was used as a template (Table I). With the BRL enzyme the yield was about 1.5%. However, reverse transcription also occurred in the absence of added primer. When denatured M d s R N A was used as a template with the BRL enzyme oligo(dG) was the least effective primer (see below). The average yield of c D N A with the other primers was 2.25%. Again, reverse transcription occurred in the absence of added primer. The molecular sizes of the reverse transcripts of L and M d s R N A were determined by alkaline agarose gel electrophoresis (Fig. 3A). With oligo(dT) as a primer numerous reverse transcripts were produced with the largest being 4.3 kb. High molecular weight reverse transcripts are also seen with oligo(dA), oligo(dC) and, especially, oligo(dG). It is unlikely that these results represent transcription of contaminating ribosomal R N A because yeast 25 S r R N A is only 3300 [30] nucleotides in length. For M dsRNA, oligo(dT) primed the synthesis of a 1.1 kb transcript and oligo(dA) primed a 1.0 kb transcript. This result is interesting because M d s R N A has been reported to contain a 190 bp region that is essentially 100% A + U [4]. This region subdivides the molecule into 1000 and 630 bp regions. The 1000 bp region encodes the killer toxin [5]. Oligo(dG) did not prime the reverse

A

kb i.4-

N.P

T

N~P.

B

4,3-

kb 1.4--

Fig. 3. A. Alkaline agarose gel electrophoresis of reverse transcripts of denatured double-stranded RNA (BRL enzyme). Lanes 1-4: oligo(dT), oligo(dA), oligo(dG) and oligo(dC) were added to reverse transcriptions of denatured L dsRNA. Lanes 5-8: oligo(dT)-, oligo(dA)-, oligo(dG)- and oligo(dC)-primed reverse transcription of denatured M dsRNA. The markers are labeled EcoRI fragments of YR p7. B. Alkaline agarose gel electrophoresisof reverse transcripts of denatured dsRNA (BRL enzyme). The gel was dried prior to autoradiography. Lane 1: 5'-end labeled EcoRl fragments of plasmid YR p7. Lane 2: denatured M dsRNA without added primer. Lane 3: oligo(dT)-primed reverse transcription of denatured M dsRNA. Lane 4: denatured L dsRNA without added primer.

transcription of high molecular weight c D N A (Fig. 3A). Oligo(dC) primed the synthesis of a 1.0 kb transcript. This probably is the result of a contaminating single-stranded R N A because oligo(dC) was found to prime reverse transcription of native

128

M dsRNA samples (29000 cpm under the conditions described in Tabel I). Reverse transcription of denatured L and M dsRNAs occurred in the absence of added primer (Table I). For L dsRNA, reverse transcription in the absence of added primer produced a 4.3 kb cDNA (Fig. 3B). Reverse transcription of M dsRNA in the absence of primer produced a 1.0 kb cDNA. The amount of high molecular weight cDNA synthesized without added primer is less than that with added primer (Fig. 3B). The size of the cDNAs synthesized with denatured M dsRNA, with oligo(dT) and oligo(dA) as primers is different (Fig. 3A). This suggests that the reverse transcripts produced with oligo(dT) and oligo(dA) are primer specific.

Hybridization of the oligo(dT) and oligo(dA)-primed cDNAs of M dsRNA to S I-cleaved M dsRNA In order to localize the segment of the M dsRNA that was reverse transcribed by oligo(dT) and oligo(dA), purified M dsRNA was digested with

A

B

Solution hybridization of the oligo(dT)- and oligo(dA)-primed cDNAs of M dsRNA In order to investigate this question, oligo(dT)and oligo(dA)-primed transcripts of M dsRNA were hybridized in solution and digested with S t

T -4-,9

L-

M-

Q

O

-1.8 kbp -

t

M2-

1 2

S] nuclease under partially denaturing conditions [5]. This results in digestion of the A + U region and cleavage of the molecule into 980 and 660 bp regions designated M1 and M2, respectively [5]. The fragments were separated by electrophoresis (Fig. 4A) and transferred to DBM-paper. Oligo(dT)- and oligo(dA)-primed reverse transcripts of M were then hybridized separately to the immobilized fragments (Fig. 4B). Both the oligo(dT)- and oligo(dA)-primed cDNAs hybridized to the M1 fragment. Both hybridized to M dsRNA marker, and neither hybridized to L dsRNA. This result shows that oligo(dT) and oligo(dA) are not priming reverse transcription of contaminating ribosomal RNA or L dsRNA (the more frequently observed contaminant). The oligo(dA)-primed cDNA hybridized to the M2 fragment as well. These results raise the question of whether the oligo(dT)- and oligo(dA)-primed cDNAs are copied from the same or different strands.

t2

0.98

-0.66

54

Fig. 4. A. Agarose gel electrophoresis of 2.5 #g each of native L and M d s R N A (lane 1) and 5 ~tg of St-digested M d s R N A (lane 2). B. Northern hybridization of the gels in A. Lanes 1 and 2: The oligo(dT)-primed c D N A of M d s R N A was hybridized following alkali digestion and G-100 chromatography. Lanes 3 and 4: The oligo(dA)-primed c D N A of M d s R N A was hybridized following alkali digestion and G-100 chromatography. The BRL enzyme was used. The approximate specific activity of the probes was 10 6 cpm//xg.

T A B L E II HYBRIDIZATION OF THE OLIGO(dT)O L I G O ( d A ) - P R I M E D c D N A s OF M d s R N A

AND

Reverse transcription of denatured M d s R N A was performed as described using the BRL enzyme. 400 c p m of the oligo{dT)primed c D N A was hybridized alone in a final volume of 20/.tl (approx. spec. act. l06 c p m / # g ) . 400 cpm of the oligo(dA)primed c D N A was hybridized alone in a total volume of 20/LI (approx. spec. act. l06 cpm/,ttg). 400 cpm of the oligo(dT)primed c D N A and 400 cpm of the oligo(dA)-primed c D N A were hybridized together in a total volume of 20 btl. Hybridization was for 40 h at 42°C. Aliquots of the hybridized samples were incubated with and without S 1 nuclease and precipitated with 5% trichloroacetic acid and counted as described. S] treatment

oligo(dT) (cpm)

oligo(dA) (cpm)

oligo(dT)+ oligo(dA) (cpm)

+ SI - S1 % S] resistant

54 106 51

24 88 27

127 196 65

129

nuclease (Table II). Prior to hybridization the transcripts were purified by Sephadex G-100 chromatography which resulted in a nonspecific loss of activity. Consequently, the values in Table II are low. Under these conditions the oligo(dT)-primed transcript exhibited self-annealing. The oligo(dA) transcript did not self-anneal. The oligo(dT)- and oligo(dA)-primed cDNAs were able to anneal to each other. The amount of S~-resistant hybrid is more than would be expected for self-annealing alone since equal amounts (cpm) of the two cDNAs were used. Discussion The dependence of reverse transcriptase on a suitable primer is well established [7-14]. However, the ability of the four oligonucleotide primers olido(dT), oligo(dA), oligo(dG) and oligo(dC) to serve as primers has been the subject of controversy. High efficiency priming by oligo(dC) was an early observation of Spiegelman et al. [31]. In this investigation addition of oligo(dT) to reverse transcription reactions of denatured L dsRNA produced a 4.3 kb reverse transcript. Oligo(dT) and oligo(dA) primed 1.1 and 1.0 kb reverse transcripts of denatured M dsRNA, respectively. Reverse transcription of L and M dsRNA also occurred in the absence of added primer. Oligo(dC) was found to be a very efficient primer for reverse transcription of yeast 18 S ribosomal RNA. The 1.0 kb reverse transcript of 18 S rRNA primed by oligo(dC) represents 59% of the length of the 1.7 kb yeast 18 S rRNA molecule. This raises the question of the location of the priming site on the 18 S rRNA molecule. It is possible that initiation occurs 1.0 kb from the 5'-end and termination occurs when the enzyme reaches the 5'-end of the molecule. This hypothesis is supported by the fact that the labeled cDNA of 18 S rRNA failed to hybridize to EcoRI fragment D, because this fragment originates from the 3'-end of the 18 S RNA gene [15]. However, this result could be due to poor transfer of the smaller EcoRI fragments to nitrocellulose. Another possibility is that initiation occurs near the 3'-end of the molecule. This possibility is plausible because this region of the molecule possesses the longest sequence devoid of secondary

structure [32] and a high content of guanine residues [33]. In this case initiation would occur near the 3'-end and proceed 1.0 kb before terminating. Termination could be the result of a kinetic barrier such as secondary structure or methylated bases. Reverse transcriptase has been shown to pause at methylated bases on the 18 S rRNA of eukaryotes [11] and the 16 S rRNA of E. coli [34]. Yeast 18 S rRNA contains six methylated bases [30]; however, their location within the molecule is unknown. Localization of the oligo(dC) priming site will require sequence analysis of the reverse transcript followed by comparison with the published sequence [3]. Sullivan et al. [27] observed reverse transcription of the large ribosomal RNA of several eukaryotic species in the absence of exogenously added primer. These authors found that the kinetics of reverse transcription of rRNA in the absence of added primer correlated with the presence of a ribonuclease activity contaminating their enzyme preparation. They proposed that this ribonuclease could uncover primers during the course of the reaction. We have not observed contaminating ribonuclease activity in the BRL enzyme. Sullivan et al. [27] suggested that priming might also be done by 5.8 S RNA molecules which are known to be hydrogen bonded to the 25 S rRNA of yeast [35]. This hypothesis is supported by our finding that reverse transcription of yeast 25 S rRNA, but not 18 S rRNA, occurs in the absence of added primer. The ability of reverse transcriptase to transcribe ribosomal RNA in the absence of added primer presents a problem when cDNAs are being synthesized for cloning purposes. Since rRNA is a frequent contaminant of poly(A)-enriched ssRNA preparations, it would be advantageous to test poly(A)-enriched preparations for the presence of minute amounts of rRNA. The efficient priming activity of oligo(dC) on ribosomal RNA may provide such a test because oligo(dC) primed the reverse transcription of ribosomal RNA contaminating the M dsRNA (Fig. 3A). The presence of ribosomal RNA in poly(A)-enriched ssRNA preparations could be easily detected because oligo(dC) primed a cDNA of distinct size when used as a primer for reverse transcription of poly(A) + RNA (Fig. 1B).

130

TOX IN 5' pppG

3' HO

TTTTTT,

s' AAAAAA AAAAA A cuuuuu.uuucuuuAc

~- 3'

M-~ 1000

' CAOH 3'

AAAA

I~-A+U 2OO

---I

, Gppp 5'

M-2 660

Fig. 5, A model of the M dsRNA genome from Ref. 5. TYhe hypothetical priming sites of the oligo(dT)- and oligo(dA)-primed reverse transcripts are indicated.

A possible initiation site for the oligo(dT)- and oligo(dA)-primed c D N A s of the M d s R N A is the region of A + U. A possible site of initiation for the oligo(dA)-primed c D N A is the sequence HOAC-U-U-U-U-U-AoU-U-U-C-U-U-U-A-C-U-G-C found at the 3'-terminus of the M1 segment, but not at the other 3'-terminus of the molecule [36,37]. This sequence has been proposed as a recognition site for the RNA-dependent R N A polymerase associated with the virus-like particles which contain the dsRNAs [36]. The oligo(dT)-primed c D N A of M d s R N A is 1.1 kb and the oligo(dA)-primed c D N A is 1.0 kb. The oligo(dT)-primed c D N A hybridized to the M1 fragment of the M dsRNA. The oligo(dA)-primed c D N A hybridized to both the M1 and the M2 fragments. The oligo(dA)- and oligo(dT)-primed c D N A s were able to hybridize to each other as well (Table II). A model for the oligo(dT)- and oligo(dA)primed reverse transcription of the M d s R N A is presented in Fig. 5. The 1.1 kb oligo(dT)-primed c D N A could be initiated in the region of A + U and reverse transcription would proceed in the 5 ' ~ 3' direction until the 5'-terminus of the M1 fragment is reached. The 1.0 kb oligo(dA)-primed c D N A could be initiated at the 3'-terminal sequence H o A - C - U - U - U - U - U - A - U - U - U - C - U - U - U A-C-U-G-C. Reverse transcription would proceed until the region of A + U is reached. A small oligo(dA)-primed c D N A could be initiated in the region of A + U on the same strand as the 1.0 kb transcript. This would account for the hybridiza-

tion of the oligo(dA)-primed c D N A products to the M2 fragment (Fig. 4B). Reverse transcriptase has been reported to be unable to use oligo(dA) as a primer when the random copolymer poly(rI,rU) is used as a template [38]. This is thought to be due to the instability of d A : r U complexes [39]. However, under our conditions oligo(dA) is capable of priming a 1000 base c D N A of denatured M dsRNA. It may be useful to note that we have found that the order of addition of reagents to the reverse transcription influences the results obtained. The oligo(dA)primed reverse transcription occurs only when the primer and R N A are added to the 5 × concentrated salt solution prior to the addition of distilled H 2 0 . This suggests that the interaction of oligo(dA) and denatured d s R N A may be dependent on a high initial salt concentration. The instability of d A : r U duplexes has been proposed as a mechanism for termination of transcription [40]. This could cause termination of the 1.0 kb oligo(dA)-primed c D N A in the region of A + U. Additional support for this hypothesis is provided by the fact that an in vivo transcript of the M dsRNA, about 1.2 kb in size, has been identified (Bostian, K.A., unpublished data). Elucidation of the precise priming sites for oligo(dT) and oligo(dA) will require sequence analysis. The M1 region of the M d s R N A has been shown to encode the killer toxin [5]. Synthesis of a double-stranded c D N A copy of this region will

131 a l l o w c l o n i n g of a ds c D N A c o p y of the d s R N A e n c o d e d toxin. A n n e a l i n g t h e o l i g o ( d T ) - a n d oligo(dA)-primed transcripts may provide a means of constructing such a double-stranded cDNA. T h e 4.3 kb c D N A o f L d s R N A p r i m e d b y o l i g o ( d T ) o f f e r s a m e a n s of o b t a i n i n g a c D N A c o p y of the L d s R N A as well. I n this case s e c o n d - s t r a n d s y n t h e sis w o u l d h a v e to b e a c c o m p l i s h e d b y the tradit i o n a l m e c h a n i s m u s i n g D N A p o l y m e r a s e I or reverse transcriptase.

Acknowledgements W e t h a n k D r . J o s e p h B e a r d for the gift of A M V r e v e r s e t r a n s c r i p t a s e . W e are i n d e b t e d to B e t h e s d a R e s e a r c h L a b o r a t o r i e s for p r o v i d i n g reverse transcriptase, restriction enzymes, S n u c l e a s e , a n d T a p o l y n u c l e o t i d e kinase. W e are g r a t e f u l to D r . Bill M a r z l u f f for the g e n e r o u s gift o f [ a - 3 2 P ] d C T P a n d to D r . D o n S i t t m a n a n d Dr. A1 M c G r a w for d i s c u s s i o n of the results p r i o r to p u b l i c a t i o n . S p e c i a l t h a n k s are d u e to L i n d a M a t t h e w s for t y p i n g the revised m a n u s c r i p t . T h i s res e a r c h was s u p p o r t e d in p a r t b y a c o n t r a c t w i t h O r g a n o n P h a r m a c e u t i c a l s I n t e r n a t i o n a l , Oss, T h e Netherlands.

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