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An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli
217
Gene, 16 (1981) 217-225 Elsevier/North-HoBand Biomedical Press
An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli (Recombinant transcription
DNA; avian retrovirus promoter; of tetracycline
and neomycin
restriction
endonucleases;
gene expression;
RNA polymerase;
resistance genes)
S. Alex Mitsialis, James F. Young *, Peter Palese * and Ramareddy
V. Guntaka
**
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, and *Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029 (U.S.A.)
(Received July 30th, 1981) (Accepted October lOth, 1981)
SUMMARY A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter active in Escherichzizcoli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA. Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promoters was shown to be dependent on the presence of an intact sequence ranging from nucleotide +I9 to -23 (relative to the cap site) in the ATV DNA. Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.
INTRODUCI’ION RNA tumor viruses contain a region in their genome, generally referred to as the common or C region, which is highly conserved among related groups of retroviruses (Wang, 1978). When the genomic RNA is converted into duplex DNA in avian tumor virus-infected cells, a sequence of about 350 nucleotides derived from the 5’ end (100 nucleotides)
** To whom correspondence should be sent, at the first address. Abbreviations: Ap, ampicillin; ATV, avian tumor virus; kb, kilobase pairs; Km, kanamycin; LTR, large terminal repeat; Nm, neomycin; Tc, tetracycline. 0378-l 119/81/0000-0000/$02.75
and the 3’ end (250 nucleotides) is duplicated to give rise to two LTRs (Hsu et al., 1978; Shank et al., 1978). In vitro transcription experiments with crude cell-free lysate systems have indicated the presence of a promoter for eukaryotic RNA polymerase II in this LTR (Yamamoto et al., 1980; S.A. Mitsialis, J.L. Manley and R.V. Guntaka, manuscript in preparation). Recently we have presented evidence for the presence of a promoter in the same region that can also be recognized and utilized by E. coli RNA polymerase (Guntaka et al., 1980; Guntaka and Mitsialis, 1980). In the present study, employing drug-resistance as a selectable marker, the promoter sequence recognized by E. coli RNA polymerase has been further
0 1981 Elsevier/North-Holland Biomedical Press
218
characterized. For this purpose, small viral DNA fragments derived from the LTRs of the Prague C strain of Rous sarcoma virus have been inserted into pBR322 in place of the TcR and Nmn gene promoters
and the recombinants
Analysis
of these recombinants
deduce the sequence
have been
isolated.
has permitted
that promotes
us to
transcription
in
E. coli.
resulting DNA was successively cleaved with BamHI and PvuII (step C). The ends of the 2.6kb fragment cont~ning the replication origin and Ap resistance gene were filled in again using deoxyribonucleotide triphosphates and avian myeloblastosis virus reverse transcriptase (Baez et al., 1980). A second copy of the 0.35kb HaeIII fragment was then inserted by blunt-end ligation to generate pATV-6D3 (step D). From this recombinant, structed.
deletion
mutants
and treated with E, coli exonuclease digestion
MATERIALS AND METHODS
(a) Bacterial strains Unless otherwise specified, E, coli HBlOl leu thi laCY StrR rk- mk- ends- red-) the recipient host in transformation routine growth, cells were grown in LB taining either 2.50 fig/ml Ap, 25 pg/ml Tc Nm.
(F-pro
was used as assays. For or LB conor 50 fig/ml
(b) Sub-cloning of viral DNA fragments in pBR322 A 1.2-kb Sac1 fragment containing both LTRs was isolated from pATV-6 (Guntaka and Mitsialis, 1980) by agarose gel electrophoresis and electroelution. This fragment was digested with HaeIII or A/u1 according to reaction conditions specified by the supplier (BRL or Boehringer-Makes) and the resulting products were inserted into pBR322. Supercoiled pBR322 DNA was successively digested with EcoRI and HindIII, the ends were filled, mixed with viral DNA fragments
were con-
pATV-6D3 DNA was linearized with BamHI with single-strand
III followed by
specific Sl nuclease
to
remove the single-strand tails (Roberts et al., 1979). BamHI linkers (Collaborative Research, Waltham, MA) were ligated to the blunt-ended fragments which were cleaved with BamHI and the plasmid was then circularized by T4 DNA ligase to regenerate the BamHI site (Baez et al., 1980) (step E). The DNA was used to transform E. coli (3300. DNA was isolated from several transformants and sequenced (Maxam and Gilbert, 1980) to determine the nucleotides removed from the WaeIII fragment by the exonuclease III treatment. (d) Insertion of the n~rnyc~
fragment into recom-
binant plasmids pKC56 DNA (Rao and Rogers, 1979) containing the NmR gene was digested with Bg/II and BamHI. A fragment of about 1.45 kb containing the NmR gene without its promoter (Jorgensen et al., 1980) was isolated and inserted at the BamHI site of pATV6D3 and its derivatives (Fig. 4) (step F). The ligated DNA was used to transform E. coli HBlOl.
and ligated by T4 DNA ligase essentially as
described by Bolivar et al. (1977). This DNA was used to transform E. co& HBlOl. The general methodology and characterization of different recombinants have been described (Guntaka and Mitsialis, 1980). (c) Construction of r~rnb~~ts the 0.35-kb HaeIII fragment
with deletions in
Supercoiled pBR322 DNA was digested with EcoRI and BamHI and the 4.25-kb large fragment was f&ed in with deoxy~bonucleotide triphosphates (Fig. 4, step A). An HaeIII fragment (0.35 kb) from the LTR was blunt-end ligated to the pBR322 (step B). This procedure regenerated the BamHI site. The
RESULTS
The general strategy for inserting WaeIII fragments of viral DNA into pBR322 is shown diagrammatically in Fig. 1. E. coli HBl 01 was transfo~ed with these recombinant molecules and the transform~ts were selected on Ap-containing agar plates. About 360 colonies were screened for Tc resistance. In a typical experiment, about 45% of the clones were Tc resistant and were shown by restriction enzyme analysis and blot hybridization to contain either the 0.35 or 0.19.kb HaeIII fragment (data not shown, see Fig. 2 for map positions). The results indicate that both of these LTR DNA fragments contain a promoter which
PvuI HoclU
Haem
Sac1
EcoRI AluI noem Hacrn
EcoRI
pATV-6.49 DNA
4
POL IKlenowJ
EcoRI
&ZTP
Hoe m
AluI Pst
pATV-6.13
A
AluIEcoRI Hoem PvuIAluIHocllI pATV -6.9
I EcoRI
pATV-6.14
AluI PvuI AhI I I 1 I EcoR I
AluI
pATV-6.91 Fig. 1. Schematic illustration for the replacement of the TcR gene promoter in pBR322 by viral DNA fragments. pBR322 DNA was doubledigested with EcoRI and Hind111 and then precipitated with ethanol. The DNA was fiied in with deoxyribonucleotides using DNA polymerase I, mixed with viral DNA fragments, generated by cleaving the 1.2-kb Sac1 fragment with Hire111 or AluI, and ligated with T4 DNA Iigase. The ligated DNA was used to transform E. coli HBlOl.
is active in transcribing the adjacent TcR gene. In the control experiment, without viral DNA fragments added, all transformants in which the 25-bp EcoRIHind111 fragment was deleted were Tc-sensitive. To test whether the orientation of the viral fragments was important for the expression of the TcR gene, several recombinants from TcR as well as TcS classes were examined for the direction of their viral DNA insert. Fig. 2 shows the orientation of different Hue111 fragments from representative recombinant clones. For example, pATV-6.49 contains the 0.19-kb fragment in the right orientation and is TcR, whereas pATV-6.10 contains the same 0.19-kb fragment in the opposite orientation and is TcS (Fig. 3). Analysis of different recombinants for growth at various Tc concentrations indicated that pATV-6.49 grew well up to 50 pg/ml, whereas pATV-6.10 failed to grow even at 2 lg/ml Tc (Table I). Likewise, pATV-6.9, which contains the 0.35-kb f&e111 fragment, also grew well up to 50 pg/ml Tc.
Horll
I EcoRI
Fig. 2. Viral DNA inserts in different recombinant clones. The 1.2-kb Sac1 fragment from pATV-6.0 (Guntaka and MitsiaIis, 1980) was digested with Hue111 or Ah1 and the resulting fragments were inserted into the EcoRI-Hind111 window of pBR322 as outlined in Fig. 1. The solid boxes represent the strong stop DNA sequence of ATV DNA. This region corresponds to the 100 $-terminal nucleotides of the ATV DNA. The orientation of the inserts in the different clones is such that the coding sequence for Tc resistance is to right of the insert.
Since the inserts
in pATV-6.49
and pATV-6.9
share a 0.14-kb sequence at the right end, these results indicate that the promoter activity resides in this 0.14 kb region, This conclusion is reinforced by the evidence obtained with pATV-6.91, in which the sequence to the left of the EcoRI site of the insert in pATV-6.9 is deleted (Fig. 2). This recombinant was found to grow at levels comparable to that of pATV6.9 (Table I). However, since there is a considerable lag in the growth of pATV-6.91 compared to pATV6.9 (Fig. 3), there may be other regulatory signals at or around the EcoRI site that are required for optimal expression. To define further the promoter sequence, the 1.2kb Sac1 fragment was digested with AluI and the resulting fragments (Fig. 2) were inserted into pBR322 as described above for the HaeIII fragments.
TABLE I Growth of various recombinants cycline
in the presence of tetra-
Growth of different recombinants in LB containing tetracycline. Hoe111 or AluI fragments of viral DNA were inserted upstream from the TcR gene as described in Methods. Overnight growth in Tc medium was recorded. Clones pATV-6.49 and pATV-6.10 have the same HaelI fragment but in reverse orientation. Likewise, pATV-6.13 and pATV6.15 have the same 190-bp A/u1 fragment in different orientations. pATV6.14 and pATV-6.16 have the same 0.26-kb AIuI fragment in opposite orientations. In the pBR332ARH clone, the 25-bp fragment between the EcoRI and Hind111 sites of pBR322 was removed.
HOURS Fig. 3. Growth curves of various recombinants in LB + Tc medium. Overnight cultures grown in LB containing Ap were diluted into fresh LB containing 10 pg/ml Tc and at various times the absorbance was determined. It should be noted that the growth of bacteria containing pATV-6.91 was delayed. At 16 h, however, the cultures reached levels similar to those of other TcR bacteria.
Results of the growth of AM recombinants indicate that both pATV-6.13 and pATV-6.14 containing the 0.19 and 0.26-kb AluI fragments, respectively, are sensitive to Tc when inserted in the same direction as in pATV-6.49 (Table I). This finding suggests that AluI cleavage interrupts the promoter activity (Fig. 5). Two clones (pATV-6.15 and pATV-6.16) containing the 0.19 and 0.26-kb AZuI fragments in the orientation opposite to that in pATV-6.13 and pATV-6.14, respectively, were also examined. As expected, pATV-6.16 was TcS but, surprisingly, pATV-6.15 grew in the presence of 20 pg/ml Tc (Table I). The reason for this unexpected observation is not known. A plausible explanation could be the presence in pATVd.15 of another sequence that can act as a promoter. Examination of the 0.19-kb AZuI fragment in pATV-6.15 reveals the presence of a sequence resembling the Pribnow box at positions -23 to -17 upstream from the junction with pBR322 (Fig. 5, nucleotides between +2 and +lO). The promoter in this clone, however, is probably a weak one since pATV-6.15 does not grow at 50 pg/ ml Tc. Since resistance to Tc appears to be a complex phenotype involving many different proteins coded by a single gene (Tait and Boyer, 1978) we have also
Clone number
pATV-6.49 pATV-6.10 pATV-6.14 pATV-6.16 pATV-6.13 pATV-6.15 p ATVd .9 pATV-6.91 pBR322 pBR322aRH
Growth in LB (Tc concentration 20
50
+ _ _ _ + + + + _
+ -
in pg/ml)
+ + + -
(+) Indicates growth; (-) indicates absence of growth.
carried out experiments with a gene conferring Nm resistance. This resistance is mediated by a single polypeptide, 3’0phosphotransferase, a structural gene product of the NmR gene carried by transposon Tn.5 (Davies and Smith, 1979). Previous studies have shown that insertion of foreign DNA at the BglII site within the NmR gene of Tn5 abolishes resistance of bacteria to the drug (Jorgenson et al., 1979). Followup experiments have clearly demonstrated that this cleavage with BglIE separates the promoter from the coding sequence of the protein conferring resistance to Nm (Rothstein and Reznikoff, 1981). Rao and Rogers (1979) constructed a pBR322 derivative (pKC7) containing the 1.8-kb HindIII-BumHI frag mcnt of the NmR gene from Tn5. Subsequently the same group derived another recombinant (pKC56) in which a BglII-Hind111 fragment of about 0.3 kb was replaced by a fragment from phage X (R.N. Rao, personal communication), thus rendering the plasmid-
221
ECORI
ECORI
BOrnHI
0 0
bearing bacterium
BOrnHI
SO11
of the promoter
sot1
BglII-BarnHI
fragment
from pKC.56 which encodes
the polypeptide that confers resistance to Nm. This fragment was then inserted into the BumHI site of plasmid recombinant pATV-6D3 and its derivatives,
7
6””
sensitive to Nm due to the removal by BglII. We have isolated a 1.45 kb
pAlVdexo11,
II
c
pATV-6exolS,
pATVdexo20
EcoR1 EcoRI BombI
pATV-6D3 has two intact HaeIII fragments, whereas other derivatives carry deletions of various sizes upstream from the Hue111 site. These deletions include nucleotide sequences ranging from +43 to +31 in exoll, +43 to t19 in exol5, +43 to t35 in exo20 and +43 to -23 in exo28 (Fig. 5). The DNAs from these recombinant plasmids containing the NmR-coding sequence and viral fragments were used to transform E. coli HBlOl. Half of the culture was plated on LB-agar plates containing 250 fig/ml Ap and the other half was plated on LB-agar plates containing 100 pg/ml Nm or Km. Transfor-
Fig. 4. Insertion of the NmR gene fragment in pBR322 carrying the viral promoter. Full details for this procedure are described in METHODS. Two 0.35-kb HaeIII fragments (cross-hatched) derived from the viral DNA were inserted in pBR322 in tandem and various deletions have been constructed from this recombinant. The 1.45kb NmR gene fragment (solid band) was inserted into each of the deletion mutants.
mants were further screened on appropriate selection media, and the DNAs were isolated from some recombinants and mapped by restriction endonucleases. Two types of transformants were detectable from colonies that grew on Ap plates. These include plasmids that are ApR and Nms and those that are resistant to both antibiotics. Colony hybridization using the 32P-labeled 1.45kb fragment of the NmR gene as a probe indicated that all the ApR NmR colonies were positive for the NmR gene. By contrast, the ApR NmS colonies included both the original plasmids as well as recombinants with the 1.45kb NmR fragment. Restriction endonuclease analysis of purified DNA from several different recombinants revealed
PAlJ-El3
G&Y UXATCGC
4.3WTAl-K
TAlTWX
&W
TACA4TA44C GCCAlTlTAC
CAllW
AllGGTGTG
CAccrrcm
~AlV6Exo2o
GMT TfXiCATCGC
/WWTAllG
TATTMTG
CCTWCE4
TACAATAAAC GCC4lTlT/X
CAlTWcAc
AlEGTGTG
CACCl-fG
PATV-ko.Ll
GAAT TCXGCATCGC /4-&3TAllG
TAlTVWG
ccTplurrcr;A TACAATPAAC GCfAlTllAC
Calls
ATlGGTGTG
C/X
14’.‘-6D(al5
GMT TUXATCGC
TAmAAGTG
CCTmM
(NDXXAC
pATv-60(028
G44T TCXZCATCGC pliw\TAIIGIBmBBG -50
and
pATV-6exo28, to test whether the viral promoter can drive expression of the NmR gene (Figs. 4 and 5).
kWb4TAlTG
-40
-30
-20
TACAATAAAC GCCAmAC
-IO
+1
+*u
+20
+30
ATI%
+40
Fig. 5. Nucleotide sequence of the viral promoter regions in pAiV-6D3 and its derivatives. The 97-bp nucleotide sequence that contains the promoter region is shown for the viral insert of pATV6D3. This region corresponds to the EcoRI-Hue111 insert in pATV-6.91 (Fig. 2). The position +1 refers to the 5’ end of virion RNA (cap site). The sequences for the deletion mutants derived from pATV-6D3 are also shown. The tentative promoter sequence for transcription in E. coli is underlined.
222 TABLE II Growth and efficiency of plating of various clones Several selected recombinants were inoculated in LB containing different concentrations of neomycin, and growth was monitored after 16 h at 37°C. Heavy growth was indicated as 4+. Efficiency of plating was determined by growing individual clones to log phase in LB containing 100 pg/ml Ap and aliquots were plated on LB agar plates containing different concentrations of Nm. The drug concentration required to reduce the number of clones by 50% was obtained graphically. Clone
pKC7
pATV6D3A pATV6D3R pATV6D3B pATV-6D32 pATVdexo1 l-3 pATV-6exoll-R pATVdexol5A pATVdexol5B pATV-exo20B pATV-exo28R
Orientation of the 1.45kb insert
Growth in LB-broth + neomycin at
EOP50
pg/mI 50
250
500
1000 fig/ml
Bg
S Ba 1
4+
4+
2+
1+
700
Bg
s
4+
4+
4+
3+
1200
P [
VP1
Ba
I
same same same same same same same same same Ba S VP1 1
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
1000
700 NT 1300 NT 700 NT 1000 20
_ Bg
pATV-D3S pATVdexollS pATV-6exolSS pATVdexo2OS pATV-6exo28S pKC56
same same same same
_ _ _ -
_ _ _ _
_ _ _ _
BU
P, Nm gene promoter; VP, viral promoter; Bg, &$I site; S, MI site; Ba, BumHI site; NT, not tested.
the presence of the 1.45 kb fragment inserted in the right orientation (viral promoter-BglII-SalI-BumHI) in all the Nmu transformants, whereas in NmS transformants it was inserted in the opposite orientation (viral promoter-BamHISaZI-BglII) (Table II). The data used to show the orientation of the 1.45-kb frag ment in a few clones are given in Fig. 6. The DNA from different recombinants was digested with PstI, which has a single site in pBR322 and two sites at 0.195 and 1 .l kb from the BglII site of the I.45kb fragment. Cleavage with PstI, therefore, should yield three fragments of 2.25, 1.30, 0.92 kb if the BglII end is adjacent to the viral promoter (viral promoterBglII-WI-BumHI). In the other orientation (viral
promoter-BarnHI-SalI-BglII) three fragments of 2.05, 1.50 and 0.92 kb are expected. Furthermore, in order to show that some PstI fragments contained both viral and NmR gene sequences, the gel was blotted to two filters and hybridized with either viral cDNA (Fig. 6, middle panel) or the 1.45 kb NmR gene fragment (Fig. 6, right panel). As expected, the 0.92-kb internal fragment contained sequences for the NmR gene only, whereas all other fragments carried both viral and NmR gene sequences. Similar results were obtained when the 1.45kb fragment was inserted into plasmids pATVdexol1, exol5 or exo20 (Fig. 6). To assess the relative
strength
of the ATV pro.
223
1234567
34567
kb
6
7
kb
6.67
-
4.26
-
2.25
L
kb
1.96’
-aI@
/ 2.25 -2.05
-
Viral
1
CDNA
\I
1
Nm Fragment (I .4!5 kb)
J
Mop of Nm Fragment Fig. 6. Blot analysis of DNA from recombinants containing the NmR gene fragment. DNAs from NmR and NmS clones were isolated and digested with PstI, and the resulting fragments were separated on a 1% agarose gel, stained with ethidium bromide and photographed under ultraviolet light (left panel). The DNA was then transferred simultaneously to two cellulose nitrate strips. One was hybridized with 32P-labeled viral EDNA (middle panel) and the other was hybridized with the nick-tr~slated 1.45kb NmR gene fragment (right panel). Note some incomplete digestion in lanes 5 and 7 of the middle panel as detected by hybridization only. HindID digest of h DNA (lane 1); PstI digest of pKC56 (lane 2); pATV-6exollS (lane 3); pATV-6exolSA (lane 4); pATV6exolSS (lane 5); pATV-6D3A (lane 6); pATV-6D3S (lane 7). pATV-6exollS, pATV-6exolSS and pATV-6D3S are NmS and pATV-exoISA and pATV-6DJA are NmR.
moter in E. coli as compared to plasmid gene promoters, several NmR clones were inoculated into L-broth containing various concentrations of Nm along with appropriate controls. The results are summarized in Table II. All NmR recomb~ants of pATV-
Model Promoter
TTG=ttt=atttgtTATnnTg=
ATV
TT ___ G ATTT-9_cGCTCGAI&CAATAA
Promoter
Fig. 7. Comparison of putative ATV promoter sequence with bacterial model promoter. The ATV promoter sequence was compared with the consensus bacterial promoter sequence taken from Rosenberg and Court (1979). In the bacterial model sequence large upper case letters are highly conserved nu~eotides, upper case letters are well conserved nucleotides, and lower case letters represent weakly conserved nucleotides. Sequences in ATV DNA in common with conserved nucleotides in the bacterial consensus sequence are underlined.
6D3, pATV8exo11, pATV-6exol5 and pATV6exo20 grew as well or better than the parental NmR plasmid, pKC7. In contrast, examination of several clones of pATV-6exo28 containing the NmR gene, in either o~entation with respect to the viral promoter, revealed only NmS transformants (Table II). This finding clearly indicates that sequences required for promotion were deleted in the pATV-6exo28 recombinants.
DISCUSSION
The results presented here strongly suggest that an ATV promoter can drive expression of plasmid genes when these genes are inserted downstream from the viral promoter. Previous experiments (Guntaka et al., 1980; Guntaka and Mitsialis, 1980) and the data
224
presented
in this paper indicate
that the promoter
recognized by E. coli RNA polymerase 50-60 nucleotide sequence upstream site (Fig. 5). Comparison
of the sequence, -1 to -30
of ATV DNA, with that of a model bacterial moter (Rosenberg
tives and to precisely map the promoter
is located in a from the cap pro-
and Court, 1979) shows surprising
homologies. The most conserved nucleotides in the bacterial consensus sequence also appear in the ATV DNA (Fig. 7). The fact that this sequence contains the promoter was supported by experiments using AZuI fragments to drive expression of the TcR gene. Since cleavage with A&I renders the recombinants Tc’, these results indicate that the promoter must reside at or around the AluI site (Fig. 5). Additional experiments using the NmR gene and the deletion mutant pATV-6exo28 indicate that a deletion extending 23 base pairs into the putative promoter sequence abolishes expression of Nm resistance. These data, therefore, suggest that the sequences on the vira.l DNA between positions -23 and i-19 (Fig. 5) are required in promoting transcription in E. coli. Although the upstream sequence that is part of the promoter has not been determined, experiments with pATV-6.91 indicate that sequences to the left of the &oRI site are not absolutely required for expression. Deletions from the 5’ end will be required to precisely define the left boundary of the promoter. Whether this prokaryotic sequence in the ATV genome is the one also recognized by eukaryotic RNA polymerase II remains an open question. Since promoter sequences for the eukaryotic enzyme are generally located in a region of -30 from the cap site (Benoist and Chambon, 1981), it is likely that the sequence located to the left of the Ai& site is the eukaryotic promoter for ATV transcription. Recently we have observed that in a eukaryotic cell-free lysate system (h4anley et al., 1979) pATV-6.13 failed to support transcription (S.A. Mitsialis, J.L. Manley and R.V. Guntaka, manuscript in prep~ation). This result can be interpreted in at least two possible ways: (1) RNA polymerase II, like E. coli RNA poiymerase, utilizes the sequence encompassing the A/u1 site and/ or (2) RNA polymerase II utilizes the sequence upstream from the A&I site but requires the downstream sequence also to rna~ta~ a certain distance from the cap site. Further in vitro transcription experiments with pATV-6exo15 and pATV-6exo28 are required to distinguish between these two alterna-
for eukaryo-
tic RNA polymerase II in ATV DNA. The observation that a promoter in ATV DNA is t
functional in E. co& and that it is very efficient has important implications and applications. Studies on the sequences required for prokaryotic and eukaryotic promoters may indicate additional homologies and contribute to a better understand~g of the evolurelationship between prokaryotic and tionary eukaryotic systems. Furthermore, the viral promoter could be used to drive expression of different genes in both prokaryotic and eukaryotic cells. For example, the Nmn gene introduced in pATV-6D3 or pATV6exo15 can be transferred into avian and other eukaryotic cells that are sensitive to G418, an analogue of Nm. Resistance to G418 can then be used as a selectable marker in transferring other genes into eukaryotic cells. Preliminary experiments using the recombinant pATV-6D3 and its derivatives containing the NmR gene have shown that G4I&resistant quail and rat cells can be selected. The expression of different genes introduced into eukaryotic cells by this selection can then be studied.
ACKNOWLEDGEMENTS
We thank Dr. R.N. Rao, Lilly Co., Indianapolis, for providing plasmids pKC7 and pRC56 and for fruitful discussions, and Miss Carol E. Smith for expert technical assistance. This work was supported by grants from the NC1 (CA28990) to R.V.G. and from NIAMID (PI% AI-1 1823), NSF (PCM 76-07848~, ACS ~V-Z3B~ to P.P. R.V.G. is a recipient of a Research Career Development award from the NCI. P.P. and J.F.Y. are the recipients of an I.T. Hirsch1 Career Scientist award and an A.A. Sinsheimer Scholar award, respectively.
REFERENCES Baez, M.,Taussig, R., Zazra, J.J., Young, J.F. and Palese, P.: Complete nucleotide sequence of the influenza A/PRJ8/ 34 virus NS gene and comparison with NS genes of the A/Udorn/72 and A/FPV/Rostock/34 strains. Nucl. Acids Res. 8 (1980) 5845-5858.
225
Benoist, C. and Chambon, P.: In vivo sequence requirements of the SV40 early promoter region. Nature 290 (1981) 304-310. Bolivar, R., Rodriguez, R.L., Betlach, M.C. and Boyer, H.W.: Construction and characterization of new cloning vehicles, 1. Ampicilhn-resistant derivatives of the plasmid pMB9. Gene 2 (1977) 75-93. Davies, J. and Smith, D.I.: Plasmid-determined resistance to antimicrobial agents. Annu. Rev. Microbial. 32 (1978) 469-518. Guntaka, R.V. and Mitsialis, S.A.: Cloning of avian tumor virus DNA fragments in plasmid pBR322: evidence for efficient transcription in E. coli from a virus-coded promoter. Gene 12 (1980) 113-121. Guntaka, R.V., Rao, P.Y., Katz, R.A. and Mitsialis, S.A.: Binding of E. coli RNA polymerase to a specific site located near the 3’-end of the avian sarcoma virus genome. Biochim. Biophys. Acta 607 (1980) 457-469. Hsu, T.W., Sabran, J.L., Mark, G.E., Gun&a, R.V. and Taylor, J.M.: Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates. J. Viral. 28 (1978) 810-818. Jorgensen, R.A., Rothstein, S.J. and Reznikoff, W.S.: Restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 1177 (1979) 65-72. Manley, J.L., Fire, A., Cano, A., Sharp, P.A. and Gefter, M.L.: DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc. Natl. Acad. Sci. USA 77 (1980) 3855-3859. Maxam, A.M. and Gilbert, W.: Sequencing end-labeled DNA with base specific chemical cleavages, in Colowick, S.P.
NOTE ADDED IN PROOF
Indeed,
we have successfully
transferred
the Nm
gene into QT6 (quail) and XC (rat) cells and found that Nm-specific RNA could be readily detected in
and Kaplan, N.O. (Eds.), Methods in Enzymology. Academic Press, New York, Vol. 65, 1980, pp. 499-560. Rao, R.N. and Rogers, S.G.: Plasmid pKC7: A vector contaming ten restriction endonuclease sites suitable for cloning DNA segments. Gene 7 (1979) 79-82. Roberts, T.M., Kacich, R. and Ptashne, M.: A general method for maximizing the expression of a cloned gene. Proc. Natl. Acad. Sci. USA 76 (1979) 760-764. Rosenberg, M. and Court, D.: Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13 (1979) 319-353. Rothstein, S.J. and Reznikoff, W.S.: The functional differences in the inverted repeats of Tn5 are caused by a single base pair nonhomology. Cell 23 (1981) 191-199. Shank, P.R., Hughes, S.H., Kung, H.J., Majors, J.E., Quintrell, N., Guntaka, R.V., Bishop, J.M. and Varmus, J.E.: Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of ciruclar DNA. Cell 15 (1978) 13831395. Tait, R.C. and Boyer, H.W.: On the nature of tetracycline resistance controlled by the plasmid pSC101. Cell 13 (1978) 73-81. Wang, L.H.: The gene order of avian RNA tumor viruses derived from biochemical analysis of deletion mutants and viral recombmants. Annu. Rev. Microbial. 32 (1978) 61-92. Y amamoto, T., de Crombrugghe, B. and Pastan, I.: Identification of a functional promoter in the long terminal repeat of Rous sarcoma virus. Cell 22 (1980) 787-797. Communicated
by Z. Hrade&&
these transformants. In addition, we have observed that continuous presence of the drug, G418, in the growth medium is not required because the transformants are stable even without selection.
Gene, 16 (1981) 217-225 Elsevier/North-HoBand Biomedical Press
An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli (Recombinant transcription
DNA; avian retrovirus promoter; of tetracycline
and neomycin
restriction
endonucleases;
gene expression;
RNA polymerase;
resistance genes)
S. Alex Mitsialis, James F. Young *, Peter Palese * and Ramareddy
V. Guntaka
**
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, and *Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029 (U.S.A.)
(Received July 30th, 1981) (Accepted October lOth, 1981)
SUMMARY A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter active in Escherichzizcoli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA. Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promoters was shown to be dependent on the presence of an intact sequence ranging from nucleotide +I9 to -23 (relative to the cap site) in the ATV DNA. Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.
INTRODUCI’ION RNA tumor viruses contain a region in their genome, generally referred to as the common or C region, which is highly conserved among related groups of retroviruses (Wang, 1978). When the genomic RNA is converted into duplex DNA in avian tumor virus-infected cells, a sequence of about 350 nucleotides derived from the 5’ end (100 nucleotides)
** To whom correspondence should be sent, at the first address. Abbreviations: Ap, ampicillin; ATV, avian tumor virus; kb, kilobase pairs; Km, kanamycin; LTR, large terminal repeat; Nm, neomycin; Tc, tetracycline. 0378-l 119/81/0000-0000/$02.75
and the 3’ end (250 nucleotides) is duplicated to give rise to two LTRs (Hsu et al., 1978; Shank et al., 1978). In vitro transcription experiments with crude cell-free lysate systems have indicated the presence of a promoter for eukaryotic RNA polymerase II in this LTR (Yamamoto et al., 1980; S.A. Mitsialis, J.L. Manley and R.V. Guntaka, manuscript in preparation). Recently we have presented evidence for the presence of a promoter in the same region that can also be recognized and utilized by E. coli RNA polymerase (Guntaka et al., 1980; Guntaka and Mitsialis, 1980). In the present study, employing drug-resistance as a selectable marker, the promoter sequence recognized by E. coli RNA polymerase has been further
0 1981 Elsevier/North-Holland Biomedical Press
218
characterized. For this purpose, small viral DNA fragments derived from the LTRs of the Prague C strain of Rous sarcoma virus have been inserted into pBR322 in place of the TcR and Nmn gene promoters
and the recombinants
Analysis
of these recombinants
deduce the sequence
have been
isolated.
has permitted
that promotes
us to
transcription
in
E. coli.
resulting DNA was successively cleaved with BamHI and PvuII (step C). The ends of the 2.6kb fragment cont~ning the replication origin and Ap resistance gene were filled in again using deoxyribonucleotide triphosphates and avian myeloblastosis virus reverse transcriptase (Baez et al., 1980). A second copy of the 0.35kb HaeIII fragment was then inserted by blunt-end ligation to generate pATV-6D3 (step D). From this recombinant, structed.
deletion
mutants
and treated with E, coli exonuclease digestion
MATERIALS AND METHODS
(a) Bacterial strains Unless otherwise specified, E, coli HBlOl leu thi laCY StrR rk- mk- ends- red-) the recipient host in transformation routine growth, cells were grown in LB taining either 2.50 fig/ml Ap, 25 pg/ml Tc Nm.
(F-pro
was used as assays. For or LB conor 50 fig/ml
(b) Sub-cloning of viral DNA fragments in pBR322 A 1.2-kb Sac1 fragment containing both LTRs was isolated from pATV-6 (Guntaka and Mitsialis, 1980) by agarose gel electrophoresis and electroelution. This fragment was digested with HaeIII or A/u1 according to reaction conditions specified by the supplier (BRL or Boehringer-Makes) and the resulting products were inserted into pBR322. Supercoiled pBR322 DNA was successively digested with EcoRI and HindIII, the ends were filled, mixed with viral DNA fragments
were con-
pATV-6D3 DNA was linearized with BamHI with single-strand
III followed by
specific Sl nuclease
to
remove the single-strand tails (Roberts et al., 1979). BamHI linkers (Collaborative Research, Waltham, MA) were ligated to the blunt-ended fragments which were cleaved with BamHI and the plasmid was then circularized by T4 DNA ligase to regenerate the BamHI site (Baez et al., 1980) (step E). The DNA was used to transform E. coli (3300. DNA was isolated from several transformants and sequenced (Maxam and Gilbert, 1980) to determine the nucleotides removed from the WaeIII fragment by the exonuclease III treatment. (d) Insertion of the n~rnyc~
fragment into recom-
binant plasmids pKC56 DNA (Rao and Rogers, 1979) containing the NmR gene was digested with Bg/II and BamHI. A fragment of about 1.45 kb containing the NmR gene without its promoter (Jorgensen et al., 1980) was isolated and inserted at the BamHI site of pATV6D3 and its derivatives (Fig. 4) (step F). The ligated DNA was used to transform E. coli HBlOl.
and ligated by T4 DNA ligase essentially as
described by Bolivar et al. (1977). This DNA was used to transform E. co& HBlOl. The general methodology and characterization of different recombinants have been described (Guntaka and Mitsialis, 1980). (c) Construction of r~rnb~~ts the 0.35-kb HaeIII fragment
with deletions in
Supercoiled pBR322 DNA was digested with EcoRI and BamHI and the 4.25-kb large fragment was f&ed in with deoxy~bonucleotide triphosphates (Fig. 4, step A). An HaeIII fragment (0.35 kb) from the LTR was blunt-end ligated to the pBR322 (step B). This procedure regenerated the BamHI site. The
RESULTS
The general strategy for inserting WaeIII fragments of viral DNA into pBR322 is shown diagrammatically in Fig. 1. E. coli HBl 01 was transfo~ed with these recombinant molecules and the transform~ts were selected on Ap-containing agar plates. About 360 colonies were screened for Tc resistance. In a typical experiment, about 45% of the clones were Tc resistant and were shown by restriction enzyme analysis and blot hybridization to contain either the 0.35 or 0.19.kb HaeIII fragment (data not shown, see Fig. 2 for map positions). The results indicate that both of these LTR DNA fragments contain a promoter which
PvuI HoclU
Haem
Sac1
EcoRI AluI noem Hacrn
EcoRI
pATV-6.49 DNA
4
POL IKlenowJ
EcoRI
&ZTP
Hoe m
AluI Pst
pATV-6.13
A
AluIEcoRI Hoem PvuIAluIHocllI pATV -6.9
I EcoRI
pATV-6.14
AluI PvuI AhI I I 1 I EcoR I
AluI
pATV-6.91 Fig. 1. Schematic illustration for the replacement of the TcR gene promoter in pBR322 by viral DNA fragments. pBR322 DNA was doubledigested with EcoRI and Hind111 and then precipitated with ethanol. The DNA was fiied in with deoxyribonucleotides using DNA polymerase I, mixed with viral DNA fragments, generated by cleaving the 1.2-kb Sac1 fragment with Hire111 or AluI, and ligated with T4 DNA Iigase. The ligated DNA was used to transform E. coli HBlOl.
is active in transcribing the adjacent TcR gene. In the control experiment, without viral DNA fragments added, all transformants in which the 25-bp EcoRIHind111 fragment was deleted were Tc-sensitive. To test whether the orientation of the viral fragments was important for the expression of the TcR gene, several recombinants from TcR as well as TcS classes were examined for the direction of their viral DNA insert. Fig. 2 shows the orientation of different Hue111 fragments from representative recombinant clones. For example, pATV-6.49 contains the 0.19-kb fragment in the right orientation and is TcR, whereas pATV-6.10 contains the same 0.19-kb fragment in the opposite orientation and is TcS (Fig. 3). Analysis of different recombinants for growth at various Tc concentrations indicated that pATV-6.49 grew well up to 50 pg/ml, whereas pATV-6.10 failed to grow even at 2 lg/ml Tc (Table I). Likewise, pATV-6.9, which contains the 0.35-kb f&e111 fragment, also grew well up to 50 pg/ml Tc.
Horll
I EcoRI
Fig. 2. Viral DNA inserts in different recombinant clones. The 1.2-kb Sac1 fragment from pATV-6.0 (Guntaka and MitsiaIis, 1980) was digested with Hue111 or Ah1 and the resulting fragments were inserted into the EcoRI-Hind111 window of pBR322 as outlined in Fig. 1. The solid boxes represent the strong stop DNA sequence of ATV DNA. This region corresponds to the 100 $-terminal nucleotides of the ATV DNA. The orientation of the inserts in the different clones is such that the coding sequence for Tc resistance is to right of the insert.
Since the inserts
in pATV-6.49
and pATV-6.9
share a 0.14-kb sequence at the right end, these results indicate that the promoter activity resides in this 0.14 kb region, This conclusion is reinforced by the evidence obtained with pATV-6.91, in which the sequence to the left of the EcoRI site of the insert in pATV-6.9 is deleted (Fig. 2). This recombinant was found to grow at levels comparable to that of pATV6.9 (Table I). However, since there is a considerable lag in the growth of pATV-6.91 compared to pATV6.9 (Fig. 3), there may be other regulatory signals at or around the EcoRI site that are required for optimal expression. To define further the promoter sequence, the 1.2kb Sac1 fragment was digested with AluI and the resulting fragments (Fig. 2) were inserted into pBR322 as described above for the HaeIII fragments.
TABLE I Growth of various recombinants cycline
in the presence of tetra-
Growth of different recombinants in LB containing tetracycline. Hoe111 or AluI fragments of viral DNA were inserted upstream from the TcR gene as described in Methods. Overnight growth in Tc medium was recorded. Clones pATV-6.49 and pATV-6.10 have the same HaelI fragment but in reverse orientation. Likewise, pATV-6.13 and pATV6.15 have the same 190-bp A/u1 fragment in different orientations. pATV6.14 and pATV-6.16 have the same 0.26-kb AIuI fragment in opposite orientations. In the pBR332ARH clone, the 25-bp fragment between the EcoRI and Hind111 sites of pBR322 was removed.
HOURS Fig. 3. Growth curves of various recombinants in LB + Tc medium. Overnight cultures grown in LB containing Ap were diluted into fresh LB containing 10 pg/ml Tc and at various times the absorbance was determined. It should be noted that the growth of bacteria containing pATV-6.91 was delayed. At 16 h, however, the cultures reached levels similar to those of other TcR bacteria.
Results of the growth of AM recombinants indicate that both pATV-6.13 and pATV-6.14 containing the 0.19 and 0.26-kb AluI fragments, respectively, are sensitive to Tc when inserted in the same direction as in pATV-6.49 (Table I). This finding suggests that AluI cleavage interrupts the promoter activity (Fig. 5). Two clones (pATV-6.15 and pATV-6.16) containing the 0.19 and 0.26-kb AZuI fragments in the orientation opposite to that in pATV-6.13 and pATV-6.14, respectively, were also examined. As expected, pATV-6.16 was TcS but, surprisingly, pATV-6.15 grew in the presence of 20 pg/ml Tc (Table I). The reason for this unexpected observation is not known. A plausible explanation could be the presence in pATVd.15 of another sequence that can act as a promoter. Examination of the 0.19-kb AZuI fragment in pATV-6.15 reveals the presence of a sequence resembling the Pribnow box at positions -23 to -17 upstream from the junction with pBR322 (Fig. 5, nucleotides between +2 and +lO). The promoter in this clone, however, is probably a weak one since pATV-6.15 does not grow at 50 pg/ ml Tc. Since resistance to Tc appears to be a complex phenotype involving many different proteins coded by a single gene (Tait and Boyer, 1978) we have also
Clone number
pATV-6.49 pATV-6.10 pATV-6.14 pATV-6.16 pATV-6.13 pATV-6.15 p ATVd .9 pATV-6.91 pBR322 pBR322aRH
Growth in LB (Tc concentration 20
50
+ _ _ _ + + + + _
+ -
in pg/ml)
+ + + -
(+) Indicates growth; (-) indicates absence of growth.
carried out experiments with a gene conferring Nm resistance. This resistance is mediated by a single polypeptide, 3’0phosphotransferase, a structural gene product of the NmR gene carried by transposon Tn.5 (Davies and Smith, 1979). Previous studies have shown that insertion of foreign DNA at the BglII site within the NmR gene of Tn5 abolishes resistance of bacteria to the drug (Jorgenson et al., 1979). Followup experiments have clearly demonstrated that this cleavage with BglIE separates the promoter from the coding sequence of the protein conferring resistance to Nm (Rothstein and Reznikoff, 1981). Rao and Rogers (1979) constructed a pBR322 derivative (pKC7) containing the 1.8-kb HindIII-BumHI frag mcnt of the NmR gene from Tn5. Subsequently the same group derived another recombinant (pKC56) in which a BglII-Hind111 fragment of about 0.3 kb was replaced by a fragment from phage X (R.N. Rao, personal communication), thus rendering the plasmid-
221
ECORI
ECORI
BOrnHI
0 0
bearing bacterium
BOrnHI
SO11
of the promoter
sot1
BglII-BarnHI
fragment
from pKC.56 which encodes
the polypeptide that confers resistance to Nm. This fragment was then inserted into the BumHI site of plasmid recombinant pATV-6D3 and its derivatives,
7
6””
sensitive to Nm due to the removal by BglII. We have isolated a 1.45 kb
pAlVdexo11,
II
c
pATV-6exolS,
pATVdexo20
EcoR1 EcoRI BombI
pATV-6D3 has two intact HaeIII fragments, whereas other derivatives carry deletions of various sizes upstream from the Hue111 site. These deletions include nucleotide sequences ranging from +43 to +31 in exoll, +43 to t19 in exol5, +43 to t35 in exo20 and +43 to -23 in exo28 (Fig. 5). The DNAs from these recombinant plasmids containing the NmR-coding sequence and viral fragments were used to transform E. coli HBlOl. Half of the culture was plated on LB-agar plates containing 250 fig/ml Ap and the other half was plated on LB-agar plates containing 100 pg/ml Nm or Km. Transfor-
Fig. 4. Insertion of the NmR gene fragment in pBR322 carrying the viral promoter. Full details for this procedure are described in METHODS. Two 0.35-kb HaeIII fragments (cross-hatched) derived from the viral DNA were inserted in pBR322 in tandem and various deletions have been constructed from this recombinant. The 1.45kb NmR gene fragment (solid band) was inserted into each of the deletion mutants.
mants were further screened on appropriate selection media, and the DNAs were isolated from some recombinants and mapped by restriction endonucleases. Two types of transformants were detectable from colonies that grew on Ap plates. These include plasmids that are ApR and Nms and those that are resistant to both antibiotics. Colony hybridization using the 32P-labeled 1.45kb fragment of the NmR gene as a probe indicated that all the ApR NmR colonies were positive for the NmR gene. By contrast, the ApR NmS colonies included both the original plasmids as well as recombinants with the 1.45kb NmR fragment. Restriction endonuclease analysis of purified DNA from several different recombinants revealed
PAlJ-El3
G&Y UXATCGC
4.3WTAl-K
TAlTWX
&W
TACA4TA44C GCCAlTlTAC
CAllW
AllGGTGTG
CAccrrcm
~AlV6Exo2o
GMT TfXiCATCGC
/WWTAllG
TATTMTG
CCTWCE4
TACAATAAAC GCC4lTlT/X
CAlTWcAc
AlEGTGTG
CACCl-fG
PATV-ko.Ll
GAAT TCXGCATCGC /4-&3TAllG
TAlTVWG
ccTplurrcr;A TACAATPAAC GCfAlTllAC
Calls
ATlGGTGTG
C/X
14’.‘-6D(al5
GMT TUXATCGC
TAmAAGTG
CCTmM
(NDXXAC
pATv-60(028
G44T TCXZCATCGC pliw\TAIIGIBmBBG -50
and
pATV-6exo28, to test whether the viral promoter can drive expression of the NmR gene (Figs. 4 and 5).
kWb4TAlTG
-40
-30
-20
TACAATAAAC GCCAmAC
-IO
+1
+*u
+20
+30
ATI%
+40
Fig. 5. Nucleotide sequence of the viral promoter regions in pAiV-6D3 and its derivatives. The 97-bp nucleotide sequence that contains the promoter region is shown for the viral insert of pATV6D3. This region corresponds to the EcoRI-Hue111 insert in pATV-6.91 (Fig. 2). The position +1 refers to the 5’ end of virion RNA (cap site). The sequences for the deletion mutants derived from pATV-6D3 are also shown. The tentative promoter sequence for transcription in E. coli is underlined.
222 TABLE II Growth and efficiency of plating of various clones Several selected recombinants were inoculated in LB containing different concentrations of neomycin, and growth was monitored after 16 h at 37°C. Heavy growth was indicated as 4+. Efficiency of plating was determined by growing individual clones to log phase in LB containing 100 pg/ml Ap and aliquots were plated on LB agar plates containing different concentrations of Nm. The drug concentration required to reduce the number of clones by 50% was obtained graphically. Clone
pKC7
pATV6D3A pATV6D3R pATV6D3B pATV-6D32 pATVdexo1 l-3 pATV-6exoll-R pATVdexol5A pATVdexol5B pATV-exo20B pATV-exo28R
Orientation of the 1.45kb insert
Growth in LB-broth + neomycin at
EOP50
pg/mI 50
250
500
1000 fig/ml
Bg
S Ba 1
4+
4+
2+
1+
700
Bg
s
4+
4+
4+
3+
1200
P [
VP1
Ba
I
same same same same same same same same same Ba S VP1 1
4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+
1000
700 NT 1300 NT 700 NT 1000 20
_ Bg
pATV-D3S pATVdexollS pATV-6exolSS pATVdexo2OS pATV-6exo28S pKC56
same same same same
_ _ _ -
_ _ _ _
_ _ _ _
BU
P, Nm gene promoter; VP, viral promoter; Bg, &$I site; S, MI site; Ba, BumHI site; NT, not tested.
the presence of the 1.45 kb fragment inserted in the right orientation (viral promoter-BglII-SalI-BumHI) in all the Nmu transformants, whereas in NmS transformants it was inserted in the opposite orientation (viral promoter-BamHISaZI-BglII) (Table II). The data used to show the orientation of the 1.45-kb frag ment in a few clones are given in Fig. 6. The DNA from different recombinants was digested with PstI, which has a single site in pBR322 and two sites at 0.195 and 1 .l kb from the BglII site of the I.45kb fragment. Cleavage with PstI, therefore, should yield three fragments of 2.25, 1.30, 0.92 kb if the BglII end is adjacent to the viral promoter (viral promoterBglII-WI-BumHI). In the other orientation (viral
promoter-BarnHI-SalI-BglII) three fragments of 2.05, 1.50 and 0.92 kb are expected. Furthermore, in order to show that some PstI fragments contained both viral and NmR gene sequences, the gel was blotted to two filters and hybridized with either viral cDNA (Fig. 6, middle panel) or the 1.45 kb NmR gene fragment (Fig. 6, right panel). As expected, the 0.92-kb internal fragment contained sequences for the NmR gene only, whereas all other fragments carried both viral and NmR gene sequences. Similar results were obtained when the 1.45kb fragment was inserted into plasmids pATVdexol1, exol5 or exo20 (Fig. 6). To assess the relative
strength
of the ATV pro.
223
1234567
34567
kb
6
7
kb
6.67
-
4.26
-
2.25
L
kb
1.96’
-aI@
/ 2.25 -2.05
-
Viral
1
CDNA
\I
1
Nm Fragment (I .4!5 kb)
J
Mop of Nm Fragment Fig. 6. Blot analysis of DNA from recombinants containing the NmR gene fragment. DNAs from NmR and NmS clones were isolated and digested with PstI, and the resulting fragments were separated on a 1% agarose gel, stained with ethidium bromide and photographed under ultraviolet light (left panel). The DNA was then transferred simultaneously to two cellulose nitrate strips. One was hybridized with 32P-labeled viral EDNA (middle panel) and the other was hybridized with the nick-tr~slated 1.45kb NmR gene fragment (right panel). Note some incomplete digestion in lanes 5 and 7 of the middle panel as detected by hybridization only. HindID digest of h DNA (lane 1); PstI digest of pKC56 (lane 2); pATV-6exollS (lane 3); pATV-6exolSA (lane 4); pATV6exolSS (lane 5); pATV-6D3A (lane 6); pATV-6D3S (lane 7). pATV-6exollS, pATV-6exolSS and pATV-6D3S are NmS and pATV-exoISA and pATV-6DJA are NmR.
moter in E. coli as compared to plasmid gene promoters, several NmR clones were inoculated into L-broth containing various concentrations of Nm along with appropriate controls. The results are summarized in Table II. All NmR recomb~ants of pATV-
Model Promoter
TTG=ttt=atttgtTATnnTg=
ATV
TT ___ G ATTT-9_cGCTCGAI&CAATAA
Promoter
Fig. 7. Comparison of putative ATV promoter sequence with bacterial model promoter. The ATV promoter sequence was compared with the consensus bacterial promoter sequence taken from Rosenberg and Court (1979). In the bacterial model sequence large upper case letters are highly conserved nu~eotides, upper case letters are well conserved nucleotides, and lower case letters represent weakly conserved nucleotides. Sequences in ATV DNA in common with conserved nucleotides in the bacterial consensus sequence are underlined.
6D3, pATV8exo11, pATV-6exol5 and pATV6exo20 grew as well or better than the parental NmR plasmid, pKC7. In contrast, examination of several clones of pATV-6exo28 containing the NmR gene, in either o~entation with respect to the viral promoter, revealed only NmS transformants (Table II). This finding clearly indicates that sequences required for promotion were deleted in the pATV-6exo28 recombinants.
DISCUSSION
The results presented here strongly suggest that an ATV promoter can drive expression of plasmid genes when these genes are inserted downstream from the viral promoter. Previous experiments (Guntaka et al., 1980; Guntaka and Mitsialis, 1980) and the data
224
presented
in this paper indicate
that the promoter
recognized by E. coli RNA polymerase 50-60 nucleotide sequence upstream site (Fig. 5). Comparison
of the sequence, -1 to -30
of ATV DNA, with that of a model bacterial moter (Rosenberg
tives and to precisely map the promoter
is located in a from the cap pro-
and Court, 1979) shows surprising
homologies. The most conserved nucleotides in the bacterial consensus sequence also appear in the ATV DNA (Fig. 7). The fact that this sequence contains the promoter was supported by experiments using AZuI fragments to drive expression of the TcR gene. Since cleavage with A&I renders the recombinants Tc’, these results indicate that the promoter must reside at or around the AluI site (Fig. 5). Additional experiments using the NmR gene and the deletion mutant pATV-6exo28 indicate that a deletion extending 23 base pairs into the putative promoter sequence abolishes expression of Nm resistance. These data, therefore, suggest that the sequences on the vira.l DNA between positions -23 and i-19 (Fig. 5) are required in promoting transcription in E. coli. Although the upstream sequence that is part of the promoter has not been determined, experiments with pATV-6.91 indicate that sequences to the left of the &oRI site are not absolutely required for expression. Deletions from the 5’ end will be required to precisely define the left boundary of the promoter. Whether this prokaryotic sequence in the ATV genome is the one also recognized by eukaryotic RNA polymerase II remains an open question. Since promoter sequences for the eukaryotic enzyme are generally located in a region of -30 from the cap site (Benoist and Chambon, 1981), it is likely that the sequence located to the left of the Ai& site is the eukaryotic promoter for ATV transcription. Recently we have observed that in a eukaryotic cell-free lysate system (h4anley et al., 1979) pATV-6.13 failed to support transcription (S.A. Mitsialis, J.L. Manley and R.V. Guntaka, manuscript in prep~ation). This result can be interpreted in at least two possible ways: (1) RNA polymerase II, like E. coli RNA poiymerase, utilizes the sequence encompassing the A/u1 site and/ or (2) RNA polymerase II utilizes the sequence upstream from the A&I site but requires the downstream sequence also to rna~ta~ a certain distance from the cap site. Further in vitro transcription experiments with pATV-6exo15 and pATV-6exo28 are required to distinguish between these two alterna-
for eukaryo-
tic RNA polymerase II in ATV DNA. The observation that a promoter in ATV DNA is t
functional in E. co& and that it is very efficient has important implications and applications. Studies on the sequences required for prokaryotic and eukaryotic promoters may indicate additional homologies and contribute to a better understand~g of the evolurelationship between prokaryotic and tionary eukaryotic systems. Furthermore, the viral promoter could be used to drive expression of different genes in both prokaryotic and eukaryotic cells. For example, the Nmn gene introduced in pATV-6D3 or pATV6exo15 can be transferred into avian and other eukaryotic cells that are sensitive to G418, an analogue of Nm. Resistance to G418 can then be used as a selectable marker in transferring other genes into eukaryotic cells. Preliminary experiments using the recombinant pATV-6D3 and its derivatives containing the NmR gene have shown that G4I&resistant quail and rat cells can be selected. The expression of different genes introduced into eukaryotic cells by this selection can then be studied.
ACKNOWLEDGEMENTS
We thank Dr. R.N. Rao, Lilly Co., Indianapolis, for providing plasmids pKC7 and pRC56 and for fruitful discussions, and Miss Carol E. Smith for expert technical assistance. This work was supported by grants from the NC1 (CA28990) to R.V.G. and from NIAMID (PI% AI-1 1823), NSF (PCM 76-07848~, ACS ~V-Z3B~ to P.P. R.V.G. is a recipient of a Research Career Development award from the NCI. P.P. and J.F.Y. are the recipients of an I.T. Hirsch1 Career Scientist award and an A.A. Sinsheimer Scholar award, respectively.
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225
Benoist, C. and Chambon, P.: In vivo sequence requirements of the SV40 early promoter region. Nature 290 (1981) 304-310. Bolivar, R., Rodriguez, R.L., Betlach, M.C. and Boyer, H.W.: Construction and characterization of new cloning vehicles, 1. Ampicilhn-resistant derivatives of the plasmid pMB9. Gene 2 (1977) 75-93. Davies, J. and Smith, D.I.: Plasmid-determined resistance to antimicrobial agents. Annu. Rev. Microbial. 32 (1978) 469-518. Guntaka, R.V. and Mitsialis, S.A.: Cloning of avian tumor virus DNA fragments in plasmid pBR322: evidence for efficient transcription in E. coli from a virus-coded promoter. Gene 12 (1980) 113-121. Guntaka, R.V., Rao, P.Y., Katz, R.A. and Mitsialis, S.A.: Binding of E. coli RNA polymerase to a specific site located near the 3’-end of the avian sarcoma virus genome. Biochim. Biophys. Acta 607 (1980) 457-469. Hsu, T.W., Sabran, J.L., Mark, G.E., Gun&a, R.V. and Taylor, J.M.: Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates. J. Viral. 28 (1978) 810-818. Jorgensen, R.A., Rothstein, S.J. and Reznikoff, W.S.: Restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 1177 (1979) 65-72. Manley, J.L., Fire, A., Cano, A., Sharp, P.A. and Gefter, M.L.: DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc. Natl. Acad. Sci. USA 77 (1980) 3855-3859. Maxam, A.M. and Gilbert, W.: Sequencing end-labeled DNA with base specific chemical cleavages, in Colowick, S.P.
NOTE ADDED IN PROOF
Indeed,
we have successfully
transferred
the Nm
gene into QT6 (quail) and XC (rat) cells and found that Nm-specific RNA could be readily detected in
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by Z. Hrade&&
these transformants. In addition, we have observed that continuous presence of the drug, G418, in the growth medium is not required because the transformants are stable even without selection.