Cloning of cDNA to a yeast viral double-stranded RNA and comparison of three viral RNAs

Gene, 19 (1982) 225-230

Elsevier Biomedical

225

Press

Cloning of cDNA to a yeast viral double-stranded RNA and comparison of three viral RNAs (Yeast killer factor;

recombinant

DNA;

yeast viral transcriptase)

Libuse A. Bobek, Jeremy A. Bruenn, Loren J. Field and Kenneth W. Gross * Division of Cell and Molecular Biology, State University of New York at Buffalo, Buffalo, NY 14260, and * Department of Molecular Biology, Roswell Park Memorial Institute, 666 Elm St., Buffalo, NY 14263 (U.S.A.) (Received

May 18th, 1982)

(Accepted

June 16th. 1982)

SUMMARY

We have constructed recombinant DNA clones containing small complementary DNA (cDNA) sequences homologous to portions of a 4.8-kb yeast viral double-stranded RNA (dsRNA) (L,) that codes for the viral capsid polypeptide. Neither the viral dsRNA nor its in vitro transcript is polyadenylated; hence the cDNAs were synthesized by reverse transcriptase on the in vitro mRNA transcript made by the viral transcriptase, using sheared salmon sperm DNA as a random primer. This is the first reported cloning of cDNA

homologous

viruses,

to a viral double-stranded

since all have a capsid-associated

RNA. This method transcriptase

activity.

should be of general The lengths

utility

for dsRNA

of the overlapping

cDNA

inserts varied from 100 to 800 bp. About 40% of them mapped to the 5’ end of the in vitro transcript, and these have been ordered. At least 1485 bp of this end of L, is represented in the cloned cDNAs characterized. Using the cloned cDNAs as probes, we have shown that the L dsRNAs are similar at the transcription initiation site and dissimilar elsewhere.

of two viral subtypes

INTRODUCTION

is complementary

The Saccharomyces cerevisiae virus (ScV) has a single molecule of genomic dsRNA (L) of about

(Welsh and Leibowitz, 1980) and can serve as an mRNA for the capsid polypeptide (Bruenn et al., 1980). The only discernible phenotype conferred

bp, base pairs;

dsRNA,

double-stranded

kilobase

pairs;

RNA;

L, 4.8 kb dsRNA

cDNA,

complementary

EtBr, ethidium

DNA:

bromide;

kb,

of ScV; M, 1.9 kb dsRNA

of

yeast satellite virus; ScV, Saccharomyces cerevisiae virus.

0378-l

1l9/82/OOOC-0000/$02.75

0 1982 Elsevier Biomedical

of the template

on infected cells is the ability to secrete a polypeptide toxin (killer toxin) if a second, satellite virus is present. The satellite virus has a single genomic dsRNA (M) of about 1.9 kb, which encodes the toxin (Bostian et al., 1980a), encapsidated in particles with the ScV capsid polypeptide (Bostian et al., 1980b). M also confers resistance to the toxin. Yeast strains with a number of killer specificities have been described (Young and Yagiu, 1978). The genomic dsRNAs of two of

4.8 kb. This dsRNA encodes the major capsid polypeptide of 88000 M, (Hopper et al., 1977). The viral particles have a capsid-associated RNA polymerase (Herring and Bevan, 1977; Hastie et al., 1978; Welsh et al., 1980) the product of which

Abbreviations:

to one strand

Press

226

these, killer types 1 and 2 (kl and k2). have been

nick-translated

compared

“pCp-labeled

by 3’ end sequencing.

ScV viral differ

dsRNAs

L,

and

We found that the L,,

of similar

at both ends but have considerable

ogy within similarly

the last 20 nucleotides. related

(Brennan

end sequence

have considerable

3’

1980). The ScV system has recently

been reviewed

The

best

remaining quire neither

methods

questions

cloned

1980). for resolving

about

cDNAs

is polyadenylated,

et al., 1981b).

RESULTS

AND

Starting transcript,

some

of the

the ScV life cycle re-

to the viral dsRNAs.

the viral dsRNA

(Brennan

In some

and Brennan,

1979; Bruenn,

et al.. 1977) or with

homol-

(Bruenn

(Wickner,

heterogeneity

(Rigby

M, and M, are

et al., 198la).

cases, the ScV viral dsRNAs

size.

probes

ScV-L, in vitro transcript

Since

nor its in vitro transcript

we have used random

stranded, tained

DISCUSSION

with 25 pg of ScV-L, we obtained dC-tailed

about

cDNA

single-stranded

60 ng of double-

for cloning.

We ob-

75 clones that were ampicillin-sensitive

tetracycline-resistant.

and

All of these hybridized

probe made by in vitro viral transcription

to a

of L, in

primed

reverse transcription of in vitro transcripts of L, to prepare a cDNA clone bank of L, sequences. We have located a number of the cloned cDNAs to the 5’ end of the transcript strand and constructed a partial map. Since the template used for reverse transcription was heavily weighted for 5’ frag-

26 45

ALU 6i pE%Rtt

53 59

21 36 48

68

ments of the transcript (Brennan et al., 198lb), our cDNA library is primarily cDNAs derived from this end of L,. This constitutes a useful set of clones for analysis of 5’ sequences of the transcript and for analysis of the sequence heterogeneity of L,. We have found that L, and L, have little sequence homology in this region, even though their capsid polypeptides are very similar (L.J. Field, L.A. Bobek, V.E. Brennan, J.D. Reilly and J.A. Bruenn,

MATERIALS

submitted).

AND

METHODS

cDNA synthesis was essentially by the method of Efstratiadis et al. (1976) using the ScV in vitro transcript (Brennan et al., 198lb) as template and an equal weight of salmon sperm DNA random primer (Taylor et al., 1976) for first strand synthesis. Double-stranded cDNA was cloned into the PstI site of pBR322 as described (Roychoudhury et al., 1976; Villa-Komaroff et al., 1978). Clones were screened (Grunstein and Hogness, 1975) with an in vitro ScV-L, transcript probe labeled with [a-32P]UTP. Hybridizations to DNA fragments or to RNA transferred to nitrocellulose were performed as described by Southern (1975) with

Fig. 1. Hybridization clones

of &I-cleaved

to ‘*pCp-labeled

transcript

DNA pause

blot). The upper figure is of the EtBr-stained used for the transfer Ah

pBR

hybridization

and hybridization

lane is AluI-cleaved control.

pBR322

from

ten cDNA

products

(Southern

gel (1.4% agarose)

in the lower figure. The DNA

as a size and

227

TABLE

I

Summary

of results of Southern

blots with nick-translated

The probes were nick-translated the horizontal question; Probe

heading,

Reaction

as in Fig. 2. ND

no detectable

with cDNA

indicates

hybridization

to the J’s11 insert.

pLl-21

pLl-26

pLl-45

+

+

-

_

_

pLl-26

-

+

+

ND

ND

pLl-21 pLI-59 pLl-11 pLl-61 pLl-2

-

+ _ _ _ _

+ + + + -

+ + + _ _

+ + + _ -

presence

of

while

[a- 32Plribonucleoside

control

was to the PstI inserts of those clones in

plus indicates

hybridization

to the Pstl

insert

in

insert

pLl-53

the

Hybridization

not determined;

pLl-45

phates,

probes

DNAs from the clones in the vertical column.

performed

minus indicates

cloned cDNA

clones

pLl-59

pLl-11

pLl-61

pLl-2

pLl-6

-

_

_

-

ND -Ii+ f +

ND _

_ _

+ + +

+ + +

+ -

triphos-

containing

only

pBR322 did not (not shown). The sizes of PstI inserts varied from about 100 bp to 800 bp (pLll-pLl-26). The GC boundaries were lo-40 bp on either end (J.A. Bruenn and G. Holmes, unpublished). The labeling script

of the CFI l-purified

of ScV-L,

in vitro tran-

with 32pCp by T4 RNA

ligase

results in the labeling of a limited set of 5’ pause products of the viral transcriptase, since these are in large molar excess over full-sized transcript. These oligonucleotides vary from 3 to about 600 nucleotides in length and originate from the 5’ end of the transcript strand (Brennan et al., 1981b). Longer products of abortive transcription are not detectably labeled (Brennan et al., 1981b). If these labeled oligonucleotides are used as a probe in Southems of Psi1 digested cloned cDNAs, the inserted PsrI fragments originating from the 5’ end of the strand

used as a template

for reverse tran-

scriptase will be the only ones to hybridize. Such an experiment is shown in Fig. 1. The insert of pLl-45 shows strong homology to the 5’ end of the transcript. The inserts of PLl-21, 53, 26, 59, 61, and 11 also show some homology, whereas those of 36, 48 and 68 do not. Of 17 tested, 7 cloned cDNAs do show some homology to the 5’ end of the transcript. This is not surprising, since the CFl 1 transcript fraction used for cDNA synthesis

Fig. 2. Hybridization clones

of PstI-cleaved

to nick-translated

DNA

The label is [a- 32P]dATP. gel used for the transfer

DNA

from pLl-61

from

six cDNA

(Southern

and hybridization

of the lower figure.

Only the PstI region of the gel is shown. The numbers clones pLl-I

I, pLl-12,

blot).

The upper figure is the EtBr-stained

etc.

refer to

22x

I !l----j 3’

L 1 TRANSCRIPT

I

5’7I , -pLi-45 I I I

----+

I I I I I

-pit-53 pLl-26

I

I

~

I I

-

I f I I I I I I I ‘-585-965bp I I I I I_ I Fig. 3. Summary

/

transcript

indicates

pLi-61

&pLi-

11 1-pLi-2 I I ---:pLl-6 I

4

I

>i485bp

of mapping from

position which

Ill_j

of cDNA

and the dashed

are derived. cDNA

to the 5’ end of the transcript.

formed

as in Fig. 2.

art’ conslatent

by Southern

Lvith

gels. Sequence

thr

map

analysih

bhow~

that the GC tails vary from 10 -40 bp (J.A. Bruenn and G. Holmes, pL1-53.

unpublished).

pLl-59.

at least

insert)+

Since the inserts ol

and pLI-61

most 5’ proximal be

portion

385 bp

length

- 160 bp (the maximum GC tails). portion

do not overlap.

the

of the pL l-6 1 insert must

(the

360 bp (the length

of the

pLI-59

of the pLl-5.3

combined

insert)

length

of the

or 585 hp from the most S’ proximal

of the ~1~1-45 insert.

Since

the labeled

I

pause products

I I I I I I

since the longest labeled pause product is estimated as about 600 nucleotides long. this places

hybridize

Probe

J

E

to the pLl-61

2

Probe

N

E

29

insert and

Probe

45

E

N

N

closest

is shown on the The dotted

line

to the 3’ end of

line the end of the pLl-6

closest

derived

patterns

clones by nick transla-

of the cDNAs

they

the end of the pLl-6

the transcript

-----+

4800b

tion. The approximate Ll

I I I I I I I I

pLi-21 pLl-59

digestion

Experiments

cDNA

were per-

has about a IO-fold excess of 5’ fragments (Brennan et al., 1981b) and, in addition, the randomprimed synthesis naturally favors copying of the 5’

E

N

E

N

half of a template RNA. We ordered the PstI inserts by a series of Southerns in which the probes were nick-translated DNAs from each of several clones and the nitrocellulose of the cloned

filters-carried DNAs.

Nick

a series of PstI digests translation

used

[a-

32P]dATP to avoid problems with the GC tails. One of the resultant Southern blots is shown in Fig. 2. A summary of these rest&s in shown in Table I. Fig. 3 gives an approximate map derived from these data and from the transcript probe data. The intensity of hybridization of the pause products to the cloned inserts roughly corresponds to their position as determined by the Southern gels (Fig. 1). Comparison of the AluI digests of some of the plasmid DNAs shows that pLl-53 and pLl-26 share two fragments of 60 and 10 bp in their cDNA inserts, and that pLl-59 and pLl-26 share one fragment of 90 bp (A&I cuts only twice within the pLl-59 insert, generating only one fragment entirely derived from the cDNA insert). The

L,-NEX-

Fig. 4. Hybridization pLl-45

DNAs

(Northern

blots). and

ScV-k,, agarose, strains

and used

T158DSK; the L,NEX results.

Total

dsRNA strains

or L dsRNA was

hybridized this

the ScV-k, strain

strains

1385. Other

were

and

from

ScV-II,. in

1.4%

transferred

to nitro-

probes

(N). The

to Nick-translated experiment

pLl-29,

ScV dsRNAs

electrophoresed

with EtBr (E), denatured, in

pLI-2.

2. 29. and 45) to various

L,NEX

stained

cellulose,

of nick-translated

(probes

the

1384, NCYC-713, k, and kz strains

ScV-k,

strain

and 1387: and gave similar

229

the most 5’ proximal

portion

the 5’ end of the transcript. size of the longest

labeled

be an underestimate,

lengths

than

Our estimate

although

about

of the pLl-45

very near

pause product

tion to the insert of pLl-2 be longer

of pLl-45

of the

may also

lack of hybridiza-

indicates

that it cannot

965 bp (the and pLl-26

sum inserts

There are at least two varieties (Brennan have

detectable

L dsRNAs

et al., a second

homology

1981a).

the replication

of the satellite virus

(L.J. Field and J.A. Bruenn,

Northern

is indistinguishable

our previous

conclusion

little homology

minus

and Brennan, hybridize

gels also confirm

that L, and M, show very

(Bruenn 1980)

unpublished

from L, by these

gels. These Northern

probes

k,, whose genomic strains

ScV-M,

data). L,Nex

of the

the GC tails).

ends

able to support

and Kane,

since none

1978; Bruenn

of the L, cDNA

to the M dsRNAs

present.

of ScV, k, and

are related at the 3’ Both

L dsRNA

k,

(L,)

and

k,

with

no

ACKNOWLEDGEMENTS

to L, (L.J. Field, L.A. Bobek,

V.E. Brennan, J.D. Reilly, and J.A. Bruenn, submitted) and with a different 3’ U-rich end (Brennan et al., 1981a). All of the mapped cDNA

We thank N. Hastie for advice and encouragement and editorial comments, W. Davidson for advice, D. Pietras for salmon sperm DNA random

inserts are submitted).

primer,

derived from L, (L.J. Field We have compared L, and

et al., L, by

“Northern” gels of the undenatured dsRNAs with nick-translated DNAs from three clones, pL l-45, 2, and 29 (Fig. 4). All of the nick-translated probes

J.W. Beard for AMV reverse transcriptase,

P. Cizdziel tance,

and

and

P. Labrozzi

the National

support (Grant No. GM19521 to K.G.).

for technical assisof Health for

Institutes

to

GM22200

J.B.

and

hybridize efficiently to L,, confirming that they all contain cDNAs synthesized on the transcript of L,. None of the nick-translated probes hybridize well to L,, although pLl-45 shows considerable homology (Fig. 4). Since the pLl-45 insert is the most 5’ proximal of the cloned cDNAs, and since L, and L, show considerable homology for at least the 5’ 27 nucleotides of the transcript (Brennan et al., 1981a), the homology to the pLl-45 insert is not surprising. The homology between L, and L, detected by the pLl-45 probe is near the transcription initiation site (Brennan et al., 1981b). Since the cDNAs of pLl-2 and pLl-29 map outside the 5’ region defined by the pCp labeled pause products, they must be at least 600 nucleotides from the 5’ end of the transcript (and from our mapping by nick translation at least 585 bp from the 5’ end of the transcript). Thus they are probably detecting differences within coding regions of L, and L,. The L, dsRNAs from three independently derived k, strains are indistinguishable by these Northern gels or by 3’ sequence analysis (Fig. 4; L.J. Field and J.A. Bruenn, unpublished data). A third viral dsRNA was also tested ogy to the three probes. This is L,Nex, polypeptide encoding dsRNA from (Wickner, 1980). This is a dsRNA with ends as L,, which, in contradistinction

for homolthe capsidstrain 1385 the same 3’ to L,, is

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