Transfer vector

A recombinant DNA vector is provided as a universal transcription vector having a replication origin and selectable marker, a promoter and a transcription initiation site comprising a first transcribed nucleotide, wherein a restriction site is provided immediately adjacent to and upstream from the transcription initiation site so as to separate transcribed from untranscribed nucleotides. A second restriction site may also be positioned downstream from the said restriction site. Precise control of initiation and termination of transcription is attained by this invention. Such control is important in assuring the effectiveness of transcribed RNA viral vectors. A high fidelity in vitro RNA transcription method is also provided utilizing vectors constructed from the universal transcription vector, or other vectors producing transcripts having no more than one extra 5' base. This method is capable of producing functional RNA transcripts, preferably comprising infectious viral sequences.

FIELD OF THE INVENTION 
This invention relates to the field of recombinant DNA and RNA vectors and 
processes for making and using same, particularly for the production of 
infectious viral RNA. 
BACKGROUND OF THE INVENTION 
The nucleotide sequences of a DNA molecule carry an information code which 
can be transcribed into an intermediate messenger (mRNA) followed by 
translation into a protein. Such a protein may have a catalytic or a 
structural function in the organism. In some cases the nucleotide sequence 
is transcribed into an RNA molecule and this RNA molecule is the 
functional entity. Natural examples are the ribosomal RNA molecules 
(rRNA), transfer RNA molecules (tRNA) and small nuclear RNA molecules 
(snRNA). Some viruses (e.g., retroviruses) are reverse transcribed and the 
DNA product is then incorporated into the host genome from whence it can 
be transcribed once again into viral RNA. A useful artificial situation 
would be to isolate a DNA fragment of exactly the same sequence as a 
useful RNA molecule and then to insert such a fragment precisely at the 
transcription initiation site downstream from a strong promoter sequence 
which in turn has been inserted into a vector plasmid. In such a case 
large quantities of the desired RNA molecule could be produced by in vitro 
transcription. Prior vector systems have provided insertion sites 
downstream from a promoter, but the RNA transcribed therefrom contains 
extraneous sequence at the 5' end. The function of the resulting RNA may 
be modified in an undesired or uncontrolled manner since many RNA 
functions are affected by the nucleotide sequence at the 5' end. This 
prior art difficulty could be overcome by a transcription vector providing 
for precise initiation of transcription of the inserted DNA segment. In 
this manner it is possible to produce wild type viral RNA sequences, 
mutated viral RNA sequences, viral RNA sequences in which some 
nonessential nucleotide sequences have been replaced by useful foreign 
genes and wild type or mutated rRNA, tRNA or sRNA molecules. Mutated viral 
RNA sequences may be used to infect plant cells thereby preventing 
superinfection or viral RNA sequences partly replaced by foreign genes may 
be used as vehicles for the introduction of those genes into plant cells. 
Alternatively the metabolism of microbial populations may be disrupted by 
transformation of the population with the recombinant plasmid carrying 
mutated rRNA, tRNA or snRNA. 
In the past several years a very large potential has developed in the field 
of genetic engineering for the transfer of foreign genes to plants. In 
order to achieve such a transfer, it is necessary to find suitable 
vectors. The most advanced work on transfer of genes to a plant genome has 
been achieved by the use of Agrobacterium tumefaciens and, to a lesser 
extent, Agrobacterium rhizogenes (Leemans, J., et al. (1982) in Kahl, G. 
and J. S. Schell (1982) Molecular Biology of Plant Tumors Ch. 21:537-545, 
Academic Press, New York). A recent study (Murai, N., et al. (1983) 
Science 222:476-482) showed that sequences coding for the bean seed 
protein phaseolin were inserted into the sunflower genome by the 
transferred DNA regions of tumor inducing plasmids. In one instance the 
phaseolin encoding sequences were controlled by the octopine synthase 
promoter and in another instance by the phaseolin promoter region. In both 
cases the phaseolin genes were correctly transcribed, processed and 
translated, thus demonstrating the expression of a plant gene after 
transfer to a taxonomically distinct botanical family. 
In addition, there is the potential of genetically engineering the normal 
plasmids of Rhizobium strains prior to the infection and formation of 
nodules on plant roots. Much work has already been done on the structure 
and function of nitrogenase genes (Scott, K. F., Rolfe, B. G. and J. Shine 
(1983) DNA 2:141-148; Scott, K. F., Rolfe, B. G. and J. Shine (1983) DNA 
2:149-156). The promoter of the nitrogenase genes of these organisms have 
been used to control the transcription and translation of a variety of 
foreign genes inserted into the symbiotic plasmids of Rhizobium in such a 
manner as to be under the control of these promoter regions (EPO 
Publication No. 0,130,047, published Jan. 2, 1985). Both types of vectors, 
i.e., the T-DNA of Agrobacterium spp. and the symbiotic plasmids of 
Rhizobium spp., involve the transfer of foreign genes as DNA. 
Another possibility for the transfer of foreign genes to plant cells is by 
the use of RNA or DNA viruses. For retroviruses, which replicate their RNA 
through DNA intermediates, naturally occurring infectious viral DNA forms 
can be isolated (J. O'Rear et al. (1980) Cell 20:423). Other RNA viruses 
replicate their genomes without passing through a DNA stage. In the case 
of these viruses, it would be necessary to reverse transcribe them into 
cDNA molecules because of the technical difficulties of handling RNA 
molecules and the lack of restriction endonuclease active on RNA. In some 
cases where amplification by secondary infection cycles is possible, e.g., 
poliovirus (Racaniello, V. and D. Baltimore (1981) Science 214:916-919) 
and potato spindle tuber virus (Cress, D., Kiefer, M. and R. A. Owens 
(1983) Nucleic Acids Res. 11:6821-6835), the method appears feasible, but 
in many examples, infectivity of viral cDNA molecules is low or 
nonexistent, and this approach does not produce results. Despite attempts 
in a number of laboratories, complete genomic cDNA clones from a range of 
other RNA viruses have not yet proved directly infectious. To understand 
why these negative results may occur, it is useful to list the following 
probable requirements for infectivity of directly-inoculated viral cDNA: 
(1) cDNA uptake into the cell; (2) cDNA transport into the nucleus; (3) 
transcription of (at least) full-length viral RNA's (vRNA); (4) avoidance 
of vRNA splicing; (5) processing of vRNA termini to infectious forms; (6) 
vRNA transport to the cytoplasm; (7) translation of (at least some) viral 
proteins; and (8) effective interaction of vRNA with viral and cellular 
proteins for a first round of replication. 
Although effective transcription is often considered to be the most 
critical feature, viral cDNA infectivity probably involves a number of 
additional requirements. For example, linkage of viral cDNA to a strong 
plant promoter might be a useful strategy but would not give infectivity 
if the viral RNA contains a cryptic splice site which leads to its 
effective processing to a non-infectious form. Also, a number of steps, 
e.g. (4) to (6) above, involve cellular processes which are poorly 
understood. Consequently, the infectivity of directly-inoculated cDNA 
could be severely limited at a number of steps, e.g., vRNA transport to 
the cytoplasm. These defects of infectivity would be extremely difficult 
to identify and so the various defects would be very difficult to correct 
without significant advances in basic molecular and cellular biology. 
Several features are needed to turn a fragment of nucleic acid into a plant 
vector: (1) The nucleic acid should be capable of being cloned so that 
useful quantities can be recovered; (2) The nucleic acid should be able to 
replicate within a plant cell and preferably, should be recognizable by 
selective methods; (3) It should not be pathogenic; (4) The nucleic acid 
must be capable of incorporating other nucleic acid sequences into its 
structure and of expressing such incorporated nucleic acid; and (5) If 
heritable changes of the transformed plant are wanted, then the vector or 
a derivative of the vector should be stably maintained from one generation 
to the next. Further, in the case of T-DNA, at some stage the transformed 
plant cell will have to be purified by cloning and regenerated into a 
plant. When these features are considered in relation to RNA plant 
viruses, a number of difficulties become apparent: (1) The techniques of 
genetically manipulating and recombining RNA are much more difficult than 
the techniques of genetically manipulating and recombining DNA; (2) Since 
most virus particles are packaged into protein capsids, there may be 
difficulties in accommodating more than limited quantities of "foreign" 
nucleic acid; (3) RNA viruses need to be replicated within the plant cell 
so there will be difficulties in recovery of genetically engineered RNA; 
(4) The strain of RNA viruses used to infect the plant cells should cause 
minimal pathogenic effects. 
The majority of known viruses infecting eukaryotes encapsidate RNA genomes. 
This is particularly true among plant viruses, where 24 of 28 recognized 
groups produce particles containing RNA (R. E. F. Matthews (1982) 
Intervirology 17:1). Cloning and manipulating cDNA copies of such viral 
RNAs has greatly facilitated progress in RNA virology in recent years. 
However, use of recombinant DNA technology in the study of most RNA 
viruses has been seriously limited by inability to express infection from 
viral cDNA clones. Methods to overcome this limitation by constructing 
complete viral cDNA clones from which infectious products can be produced 
by in vitro transcription are needed. Expression of viral cDNA by such 
methods allows detailed molecular genetic analysis of RNA virus 
replication, gene expression, and regulation and the construction of 
practical expression vectors based on RNA viruses. 
SUMMARY OF THE INVENTION 
A method of constructing a recombinant plasmid carrying a vector, a strong 
promoter and a restriction endonuclease site at the transcription 
initiation point is described. This restriction endonuclease site is used 
to insert the desired DNA fragment. 
The techniques of recombinant DNA have made it possible to transcribe and 
translate a variety of open reading frames in a DNA sequence. The 
efficiency of such transcription and translation depends on many factors, 
e.g., the "strength" of the promoter region, the structure of DNA 
sequences upstream from the transcription intitiation site, and, in some 
cases, the DNA structure on the 3' side of the termination signal. In some 
cases it is useful to produce an RNA transcript with precise initiation 
and termination points, e.g., for the in vitro production of viral RNA 
molecules, small nuclear RNA molecules and rRNA molecules. This invention 
describes the production by use of advanced genetic engineering techniques 
of a recombinant plasmid containing a restriction endonuclease site at a 
point downstream from a strong promoter, preferably a lambda virus 
promoter (PR) where transcription from various DNA sequences can be 
precisely initiated. In addition, a restriction site, preferably an EcoRI 
site, occurs immediately after the transcription initiation site so the 
use of the restriction immediately prior to transcription in vitro will 
also precisely locate the termination point (within 7 nucleotides) of any 
DNA fragment inserted into the initiation restriction endonuclease site. 
A preferred embodiment of the universal transcription vector of this 
invention is designated pPM1, and was deposited with the American Type 
Culture Collection Depository in Rockville, Maryland on March 7, 1985, as 
ATCC No. 40172. 
A preferred recombinant DNA vector of this invention comprises: (a) a 
replication origin and selectable marker; (b) a nucleotide sequence 
comprising promoter sequences and a transcription initiation site wherein 
a transcription initiation nucleotide is the first transcribed nucleotide; 
(c) a restriction endonuclease recognition sequence whose cleavage site is 
downstream from said promoter on the 3' side of the transcription 
initiation nucleotide and no more than 1 base removed therefrom. 
The promoter sequence often is modified from the natural promoter sequence 
so as to contain a portion of the restriction endonuclease recognition 
sequence. Preferably the restriction endonuclease recognition sequence is 
a blunt-end restriction endonuclease site, most preferably SmaI. 
The promoter sequence may be any promoter sequence capable of regulating 
RNA polymerase. Preferably the promoter sequence is that of pPM1, a 
P.sub.R -lambda phage promoter. Other preferred promoters are the 
T7.phi.10 and SP6 promoters, although any promoter known to the art may be 
used. Such promoters may be synthetically derived, or derived from 
organelles or nuclear regions of many organisms including bacteria, 
bacteriophages, viruses, fungi and algae, plants and animals. 
Any replication origin and selectable marker known to the art may be used, 
preferably those of pUC9 or M13mp9. 
The vectors of this invention may also comprise inserted sequences coding 
for desired RNA transcripts inserted at the above-mentioned cleavage site. 
Preferred RNA sequences are viral RNA sequences, symptomless viral RNA 
sequences, rRNa, tRNA, and snRNA, or mutated forms of the foregoing. 
In a second embodiment of this invention, the vector contains such inserted 
sequences and lacks a restriction endonuclease site, or part thereof at 
the junction between the inserted sequence and the promoter sequence; 
however, the first-transcribed nucleotide must be the first nucleotide of 
the desired RNA transcript, or must be immediately adjacent and 5' to said 
first nucleotide of the desired RNA transcript. 
The vectors of this invention may also contain an endonuclease recognition 
sequence on the 3' side of the endonuclease recognition sequence mentioned 
above, and immediately adjacent thereto. When exogenous DNA to be 
transcribed is inserted between the two recognition sequences, it is 
possible to controllably terminate transcription so that the transcript 
contains no more than seven additional bases on the 3' end. 
The vectors of this invention may be prepared by processes known to the art 
as described in the Examples hereof. A preferable method for preparing a 
universal transcription vector of this invention comprises the steps of: 
(a) isolating a DNA fragment containing a promoter; (b) inserting said DNA 
fragment into the replicative form of a filamentous single-stranded phage 
strain to produce a recombinant filamentous single-stranded phage; (c) 
transforming a bacterial strain with said recombinant phage; (d) isolating 
ssDNA of said recombinant phage excreted from said bacterial strain; (e) 
synthesizing an oligonucleotide primer partially complementary to said 
promoter, the 5' end of said oligonucleotide primer corresponding to a 
transcription initiation site and said 5' end having a sequence 
complementary to one side of a blunt-end restriction endonuclease site; 
(f) replicating said ssDNA using said oligonucleotide primer to produce a 
modified complementary DNA strand containing a modified promoter, the 
modified promoter being the promoter comprising a sequence complementary 
to said oligonucleotide primer; (g) replicating said modified 
complementary DNA strand to produce a modified double stranded DNA 
fragment containing said modified promoter; and (h) cloning said modified 
double stranded DNA fragment into a vector plasmid resulting in a 
universal transcription vector. The term "one side" as used in step (e) 
above relative to a restriction site means the side of the site containing 
all the bases on one side of the cut side. 
Any DNA fragment containing an appropriate promoter as described above may 
be used. In one embodiment the fragment is a cI-P.sub.RM -P.sub.R from 
phage lambda. Other preferred promoters are T3, T7 (.phi.10) and SP6. 
"Promoter" as used herein means sequences having the function of a 
promoter, i.e., in initiation of transcription. A promoter useful herein 
need not be the entire sequence of the promoter in its naturally-occurring 
form, but can merely be sufficient sequences of the naturally occurring 
promoter to function equivalently to the naturally-occurring promoters. 
Many means for isolating and constructing such functional promoters not 
requiring undue experimentation are known to the art and, for example, may 
involve simply testing the proposed promoter in a vector construct having 
a reporter gene. Filamentous single stranded phage strains are known to 
the art, such as M13mp9. Any bacterial strain may be used for transforming 
with the recombinant phage. Preferred bacterial strains are E. coli 
K12JM101 and 103. 
The means for constructing appropriate primers will be apparent to those 
skilled in the art. The primer used in the preferred embodiment of this 
invention is 5'-OH-d(GGGACCATTATCACC)-3'-OH, used with the preferred 
restriction site, a SmaI site. When other restriction sites are used, 
other primers may be constructed as known to the art. 
The process of making the universal transcription vectors of this invention 
also includes the insertion of DNA fragments corresponding to desired RNA 
transcripts at the restriction site. 
An in vitro process for preparing high-tolerance RNA transcripts is also 
provided by this invention. The term "high-tolerance" as used herein means 
transcript having no more than one extra base at the 5' end. It has been 
found that viral RNA transcripts having one more than one extra base at 
the 5' end are not efficiently infective to whole plants. Recombinant DNA 
vectors in which the transcription intitiation nucleotide corresponds to 
the extreme 5' nucleotide of the desired functional RNA in its natural 
state are included within the scope of this invention, as are vectors in 
which the first transcribed nucleotide is immediately adjacent to the 
nucleotide corresponding to the extreme 5' nucleotide of the desired 
functional RNA. 
Such high tolerance RNA transcripts are prepared in vitro by: 
(a) preparing a cDNA sequence corresponding to the RNA transcript desired; 
(b) providing a promoter sequence attached to said cDNA sequence capable of 
controlling transcription thereof in an in vitro environment; 
(c) replicating said cDNA sequence and attached promoter sequence; 
(d) placing replicated sequences of step (c) in an in vitro environment 
comprising an RNA polymerase regulated by said promoter sequences so as to 
permit transcription of functional RNA transcripts corresponding to said 
cDNA; 
(e) isolating functional RNA transcripts from the materials of (d). 
Useful promoters are as described above, and useful RNA polymerases such as 
those of E. coli and phages T3, T7 and SP6 are similarly known to the art, 
as are in vitro conditions for RNA transcription and suitable vectors not 
requiring nor providing the high tolerance of this invention. 
The vectors utilized for the in vitro transcription processes of this 
invention need not have a restriction site or part thereof with a cleavage 
point immediately 5' to the transcription initiation nucleotide. In other 
words, they need not be constructed using the universal transcription 
vector of this invention. All that is necessary is that the transcription 
initiation site corresponds to, or is one base upstream from, the 
nucleotide corresponding to the extreme 5' nucleotide of the functional 
RNA. 
A functional RNA as used herein means an RNA which is capable of performing 
the function its corresponding RNA performs in nature, e.g., tRNA, rRNA, 
snRNA, and preferably viral RNA. The functional RNAs may be modified so as 
to no longer perform the function intended by nature, for example viral 
RNA may be made symptomless, tRNA may be mutated, etc.; however, so long 
as the RNA transcripts are capable of substituting for the corresponding 
unmodified RNAs, they are considered functional for purposes of this 
discussion. Functional RNA's also include RNA's capable of translation to 
produce useful proteins. 
This invention also includes the functional, high-tolerance RNA transcripts 
produced by the above-described processes. 
Bacterial strains containing and replicating the above-described vectors 
are also included within the scope of this invention. Many suitable 
bacterial strains are known to the art. Preferable strains are E. coli 
K12JM101 and E. coli K12JM103.

DETAILED DESCRIPTION OF THE INVENTION 
A key feature of the present invention is the construction of recombinant 
expression vectors with strong promoter elements and restriction 
endonuclease sites positioned downstream from the promoter so that 
transcription initiation always occurs at a precise nucleotide. Another 
critical feature of the present invention is the discovery that, following 
the conversion of viral RNA molecules to cDNA by the use of reverse 
transcriptase, these cDNA molecules can be transcribed in vitro into RNA, 
and that such viral RNA molecules transcribed in vitro from cDNA molecules 
are infectious, provided that transcription, initiation and transcription 
termination are essentially at the correct nucleotides. 
To be useful in the transmission of foreign genes into plants, DNA 
fragments are incorporated into suitable positions on the cDNA molecule. 
For example, using brome mosaic virus which has a tripartite genome (i.e., 
RNA1, RNA2 and RNA3), it is possible to delete the nucleic acid sequence 
coding for the coat protein in RNA3 and replace it with the sequence 
coding for a foreign gene. Such a deletion and replacement is most easily 
done by use of the cDNA molecule. 
This strategy has several attractions: (a) Gene 3a occurs as one of the two 
open reading frames of RNA3 and it is necessary for this gene to be 
present for viral RNA replication within plant cells. The requirement for 
gene 3a function would serve to maintain the engineered component in the 
virus; (b) The foreign protein would presumably be strongly expressed by 
the same mechanisms which lead to strong coat protein production in normal 
infection; (c) The size of the inserted sequences would not be limited by 
encapsidation constraints; (d) The lack of viral coat protein should limit 
the escape of engineered virus from deliberately inoculated plants. If 
both the 3a and coat genes (encoded by RNA3) were required for infection, 
they could be separated onto two different components, one and coat genes 
(encoded by RNA3) were required for infection, they could be separated 
onto two different components, one or both of which could also carry 
foreign genetic information. Up to 2kb of foreign sequence may be inserted 
in a single such component without violating packaging constraints. 
Many other viruses are useful for the in vitro production of infectious RNA 
molecules by transcription from cloned cDNA, including various other plant 
viruses [e.g., tobacco mosaic virus (TMV)], poliovirus, influenza virus, 
foot and mouth disease virus, and others. 
There is yet another benefit to be obtained from the in vitro transcription 
of viral mRNA molecules from cDNA. It is possible to produce mutations in 
the viral cDNA molecules that convert a virulent strain to a symptomless 
strain. Such symptomless strains are used for in vitro synthesis of viral 
mRNA which is genetically engineered to contain foreign genes. Infection 
of plants by such symptomless strains of virus can also prevent later 
superinfection of these plants by virulent strains of virus. 
Another use of a recombinant vector carrying a strong promoter and allowing 
transcription start no more than one nucleotide upstream from the 
naturally-occurring RNA to which corresponding transcripts are desired is 
the use of mutated rRNA molecules to combat bacterial infections. rRNA 
molecules are transcribed from rDNA. Thus, it is possible to mutate the 3' 
end of the bacterial 16S rRNA molecule in such a way that the rRNA takes 
its place in the ribosomes but is incapable of mediating translation of 
mRNA molecules. Multiple transformation of bacteria by a recombinant 
vector containing such a mutated rRNA severely retards or eliminates 
bacterial infection. Similarly, mutated tRNA's which are transcribed from 
tDNA's could be used for the multiple transformation of bacterial 
populations. Such mutated tRNA's can cause a severe disruption of the 
bacterial capability to translate mRNA's into needed proteins. 
Yet another use of the transfer vectors described herein is the enhancement 
of translation of a mRNA transcribed from a eukaryotic gene in a 
transformed bacterium. It is well known in the art that tRNA populations 
of bacteria occur in different proportions compared to the tRNA 
populations of eukaryotes and that codon usage is different in the mRNA's 
of prokaryotes and eukaryotes. Translation of a mRNA transcribed from a 
eukaryotic gene in a transformed bacterium can be optimized by further 
transforming a variety of DNA's encoding tRNA's under the control of a 
promoter into the transformed bacterium. 
In order to clone these various DNA molecules from which precisely 
initiated and terminated ("high resolution") transcripts are required, it 
is necessary to construct a recombinant plasmid where the DNA to be 
transcribed is inserted at a precise distance downstream from the 
regulatory signals of a strong bacterial or viral promoter. Bacteriophage 
promoters frequently provide strong initiation signals because an 
infecting virus must redirect the cellular metabolism of a host to its own 
purposes. It has been shown that lambda P.sub.R promoter initiates 
transcription many times more frequently than bacterial promoters such as 
gal or lac (Queen, C. and M. Rosenberg (1981) Cell 25:241-249). 
In previous work to increase the production of proteins (Queen, C. (1983) 
J. Mol. Appl. Genet. 2:1-10), an expression vector was created by placing 
the P.sub.R promoter and cro gene ribosome binding site on a plasmid, 
along with a repressor gene for P.sub.R in such a way that the cro 
ribosome binding site could be easily linked to foreign genes. The 
effectiveness of this vector was shown by producing two proteins, 
.beta.-galactosidase and SV-40 small t-antigen, at the level of 5-10% of 
total E. coli protein. The level of t-antigen achieved (4.times.10.sup.5 
molecules/cell) was about 10 times higher than was obtained with the lac 
system (Thummel, S., Burgess, T. L. and R. Tijian (1981) J. Virol. 
37:683-697) or a chemically synthesized ribosome binding site (Jay, G., 
Khoury, G., Seth, A., and Jay, E. (1981) Proc. Nat. Acad. Sci. U.S.A. 
78:5543-5548) and is 20 times higher than the level of interferon obtained 
using a triple trp promoter (Goeddel, D. V., Shepard, H. M., Yelverton, 
E., Leung, D. and R. Crea (1980) Nucleic Acids Res. 8:4057-4073). 
This expression vector which carried the P.sub.R phage lambda promoter 
(Queen, C. (1983) J. Mol. Appl. Genet. 2:1-10) was the starting point for 
creating an entirely novel vector, namely, a vector carrying a strong 
promoter with a restriction site downstream from the regulatory sequences 
of the strong promoter precisely at the transcription initiation site. 
Since the restriction site can be used to insert foreign DNA, this means 
that transcription initiation of any foreign DNA can be precisely 
positioned. 
In the construction of these transcription initiation vectors, one is not 
confined to the use of the P.sub.R lambda phage promoter. Any suitable 
promoter may be used; such a promoter may be strong or weak and subject to 
regulation by activators and/or repressors. A representative list of such 
promoters would include (but not be restricted to) the following list: (a) 
Bacterial promoters such as galP1 (Musso, R. et al. (1977) Proc. Nat. 
Acad. Sci. U.S.A. 74:106-110) or trp (Bennett, G. N. et al. (1978) J. Mol. 
Biol. 121:113-137; (b) Other phage promoters such as T3 and T7 promoters, 
preferably the .phi.10 T7 promoter (Dunn, J. J. et al. (1983) J. Mol. 
Biol. 166:477-535; and including the T7 A1 promoter (Siebenlist, U. (1979) 
Nucleic Acids Res. 6:1895-1907), or those of phage SP6 (Green, M. et al. 
(1983) Cell 32:681-694); (c) Plasmid and transposon promoters such as 
pBR322bla (Sutcliffe, J. G. (1979) Proc. Nat. Acad. Sci. U.S.A. 
75:3737-3741) and Tn5neo (Rothstein, S. J. et al. (1981) Cell 23:191-199) 
and promoters created by fusion or mutation such as lacP115 (Johnson, R. 
C. et al. (1981) Nucleic Acids Res. 9:1873-1883) and IS2 I-II (Hinton, D. 
M. and R. E. Musso (1982) Nucleic Acids Res. 10:5015-5031). In addition, 
completely synthetic promoters or promoters recognized by RNA polymerases 
from biological sources other than bacteria may be used, e.g. fungal, 
algal, viral, plant or animal; and these promoters may be nuclear or 
organellar promoters. 
The vectors used in the present invention for the insertion, propagation 
and manipulation of the BamHI-ClaI DNA fragment from the expression vector 
pCQV2 (FIG. 1) was M13mp9 (Messing, J. and Vieira, J. (1982) Gene 
19:269-276). The M13mp9 in this example was chosen for ease and 
convenience. The use of this strain is not to be interpreted as excluding 
the use of other filamentous single stranded phages (e.g., f1 or fd). 
Other filamentous, single stranded phages could equally well be utilized 
in these experiments (The Single Stranded DNA Phages (1978) Denhardt, D. 
T., Dressler, D. and D. S. Ray, eds. Cold Springs Harbor Laboratory, New 
York). M13mp9 was derived from another M13 engineered phage, i.e., M13mp7. 
The cloning of DNA into the replicative form (RF) of M13 has been 
facilitated by a series of improvements which produced the M13mp7 cloning 
vehicle (Gronenborn, B. and J. Messing (1978) Nature, Lond. 272:375-377; 
Messing, J. (1979) Recombinant DNA Technical Bulletin, NIH Publication No. 
79-99, 2, No. 2 43-48; Messing, J., Crea, R. and P. H. Seeburg (1981) 
Nucleic Acids Res. 9:309-321). A fragment of the E. coli lac operon (the 
promoter and N-terminus of the .beta.-galactosidase gene) was inserted 
into the M13 genome. This lac promoter-operator insert codes for the first 
145 amino acid residues of the .beta.-galactosidase gene. During the 
infection of certain cell lines (e.g., E. coli K12 JM101 and JM103) this 
information will complement certain deletion mutants of the 
.beta.-galactosidase gene and restore .beta.-galactosidase activity 
(.alpha.-complementation). These cells show a lac.sup.+ phenotype and can 
be identified by a blue colored plaque on a medium containing IPTG and 
X-gal (Malamy, M. H., Fiandt, M. and Szybalski, W. (1972) Mol. Gen. Genet. 
119:207ff). In addition, a small DNA fragment synthesized in vitro and 
containing an array of restriction cleavage sites [a multiple cloning site 
(MCS)] was inserted into the structural region of the .beta.-galactosidase 
gene fragment. In spite of these insertions the M13mp7 DNA is still 
infective and the modified lac DNA is able to encode the synthesis of a 
functional .beta.-galactosidase .alpha.-peptide (Langley, K. E., 
Villarejo, M. R., Fowler, A. V., Zamenhof, P. J. and I. Zabin (1975) Proc. 
Nat. Acad. Sci. U.S.A. 72:1254-1257). This synthesized DNA fragment in 
M13mp7 contains two sites each for the EcoRI, BamHI, SalI, AccI and HincII 
restriction enzymes arranges symmetrically with respect to a centrally 
located PstI site. By chance, either strand of a cloned restriction 
fragment can become part of the viral (+) strand. This depends on the 
fragment orientation relative to the M13 genome after ligation. The 
insertion of a DNA fragment into one of these restriction sites is readily 
monitored because the insertion results in a non-functional 
.alpha.-peptide and the loss of .beta.-galactosidase activity. Under 
appropriate conditions, e.g., when strains E. coli K12 JM101 or 103 are 
infected by M13mp7, the functional .alpha.-peptide results in blue 
plaques; a non-functional .alpha.-peptide results in colorless plaques 
(Messing, J. and B. Bronenborn (1978) In Denhardt, D. T., Dressler, D. and 
D. S. Ray (eds.) The Single Stranded DNA Phages. Cold Spring Harbor 
Laboratory, Cold Spring Harbor, New York, pp. 449-453). M13mp7 has found 
wide application in the dideoxy nucleotide sequencing procedure (Sanger, 
F., Nicklen, S. and A. R. Coulsen (1977) Proc. Nat. Acad. Sci. U.S.A. 
74:5463-5467). 
A disadvantage of M13mp7 was that the multiple cloning site (MCS) contained 
two of each kind of restriction endonuclease site thus allowing DNA 
restriction fragments to be inserted in both orientations. Therefore, two 
new ssDNA bacteriophage vectors, M13mp8 and M13mp9, were constructed 
(Messing, J. and Vieira, J. (1982) Gene 19:269-276). The nucleotide 
sequence of M13mp7, containing the multiple restriction sites, was 
modified to have only one copy of each restriction site and, in addition, 
single HindIII, Sma I and XmaI sites. Thus, DNA fragments whose ends 
correspond to two of these restriction sites can be "forced cloned" by 
ligation to one of these new M13 cloning vehicles that has also been "cut" 
with the same pair of restriction enzymes. M13mp8 and M13mp9 have the 
modified multiple restriction site region arranged in opposite 
orientations relative to the M13 genome and a given restriction fragment 
can be directly oriented by forced cloning. The use of both vectors M13mp8 
and M 13mp9 guarantees that each strand of the cloned fragment will become 
the (+) strand in one or the other of the clones and thus be extruded as 
ssDNA in phage particles. 
Initially, fusion of the lambda phage P.sub.R promoter to the pUC9 
polylinker resulted in 21 nucleotides of lambda sequence and some 
additional pUC9 nucleotides appearing at the 5' end of the synthetic brome 
mosaic virus cDNA3 transcript (FIGS. 2 and 3). In order to allow the 
production of synthetic RNA transcripts (including viral RNA transcripts) 
with correct 5' termini, a "universal" transcription vector, now 
designated pPM1, was created. This transcription vector (pPM1) retained 
most elements of the P.sub.R promoter but the transcription initiation 
site coincided with the cut side of a unique blunt-end restriction site 
(the SmaI site in FIG. 3b). The construction of such a transcription 
vector became possible when examination of a large number of promoter 
sequences revealed that it might be possible to generate a blunt-end SmaI 
site at the position of transcription initiation by specific site directed 
mutagenesis without altering the promoter strength. Transcription of DNA 
fragments blunt-end ligated into this SmaI site was later demonstrated to 
begin at the first nucleotide of the inserted fragment. In particular, 
viral cDNA's were primed by terminally complementary oligonucleotides, 
blunt-end ligated into this site and transcribed to give the correct 5' 
ends. Further, the initial form of this vector (FIG. 3b) also provided a 
unique EcoRI site immediately downstream of the SmaI site where 
transcription could be runoff-terminated to give product RNA's with no 
more than seven additional nucleotides at the 3' end. 
To construct the pPM1 vector, the lambda cI-P.sub.RM -P.sub.R cartridge 
from pCQV2 (FIG. 2) was subcloned into M13mp9. The resulting ssDNA was 
used as a template for DNA synthesis from a mismatched oligonucleotide 
designed to fuse the initiation site to the left half of the SmaI 
recognition sequence. The mismatched oligonucleotide was synthesized by an 
"in-house" facility. The DNA copied from the mismatch primer was made 
double stranded, isolated and ligated into M13mp9. After verifying the 
sequence of the modified P.sub.R promoter (i.e., PM promoter), the new 
cI-P.sub.RM -P.sub.M cartridge was subcloned into pUC9 to give the final 
desired vector pPM1. Several non-trivial technical difficulties were 
encountered in the use of the mismatch primer. It was found at first that 
the primer was complementary to a nucleotide sequence which was 96 
residues upstream from the P.sub.R promoter and that successful priming 
was not achieved 3 nucleotides upstream from the P.sub.R promoter 
initiation site as was expected. 
The solution of this unexpected result was finally reached after an 
exhaustive series of experiments which eliminated other possibilities and 
showed that the initial cloning protocol failed at the stage of oligomer 
binding. A comprehensive consideration of possible mechanism of failure to 
bind oligomer at the desired site and a computer analysis of possible 
complementary pairing situations culminated in the conclusion that 
secondary structure in the template was the most likely problem (FIG. 5). 
This discovery then led to the development of a novel strategy to drive 
binding to the desired site despite competition from intra-molecular 
interactions. This novel strategy included use of reverse transcriptase in 
place of DNA polymerase I Klenow fragment (FIG. 4) and complete 
denaturation followed by slow renaturation of the template in the presence 
of a high effective concentration of the mismatched primer. Finally, 
direct of this newly developed strategy using a wide range of primer 
concentrations to determine the required levels of primer resulted in 
effective intitiation of DNA synthesis at the desired position, namely, 3 
nucleotides upstream from the P.sub.R promoter initiation site. 
The recognition sequence of the SmaI restriction endonuclease is: 
##STR1## 
The cleavage site occurs in the center between C and G. The reading strand 
is 3'-G-G-G-C-C-C-5'. The cleavage site of the SmaI restriction 
endonuclease is therefore on the 3' side of the transcription intitiation 
nucleotide with respect to the reading strand. Again, with respect to the 
reading strand, "upstream" is defined as 5'.fwdarw.3' and "downstream" is 
defined as 3'.fwdarw.5'. 
In general, the critical feature in the construction of universal 
transcription initiation vectors is the distance between the promoter 
sequence and the blunt-end cleavage site of a restriction endonuclease. 
The cleavage site of the restriction endonuclease must be on the 3' side 
of the nucleotide which is used for transcription initiation on the 
reading strand such that the cleavage site separates transcribed from 
untranscribed nucleotides. The term "transcription initiation nucleotide", 
as used herein, means the first transcribed nucleotide, i.e., the 
nucleotide corresponding to (or complementary to, depending on the DNA 
strand) the 5'-terminal ribonucleotide of the RNA transcript. 
E. coli RNA polymerase, which recognizes the P.sub.R promoter in pPM1, is 
capable of transcribing RNAs with a wide variety of 5' end sequences (D. 
Hawley and W. McClure (1983) Nucleic Acids Res. 11:2237). However, most 
natural RNAs produced by this enzyme begin with purine. A number of 
sequences starting with a 5' purine have been linked to the P.sub.R 
promoter sequences in pPM1 and in all cases tested transcription 
initiation from P.sub.R began as expected at the initial purine nucleotide 
of the inserted fragment. (P. Ahlquist and M. Janda (1984) Mol. Cell. 
Biol. 4:2876). 
The next step is to clone exact cDNA copies of the various RNA molecules, 
i.e., the three genomic RNA's of brome mosaic virus or other viruses (see 
supra), the 16S RNA of various bacteria and the snRNA's from eukaryotic 
cells. These steps can be accomplished by synthesizing oligonucleotides 
complementary to each end of the RNA for use as first and second strand 
cDNA primers. Once the cDNA's have been synthesized and cloned into the 
unique restriction site of the universal transcription vector, preferably 
the SmaI site of the novel pPM1 vector plasmid, it is then possible to 
synthesize RNA which differ from their authentic natural RNA counterparts 
only by the addition of a maximum of seven nucleotides to the 3'-end. 
Other starting vectors may be used to prepare equivalent universal 
transcription vectors to those described herein, as will be apparent to 
those skilled in the art. Similarly, other restriction sites, 
transcription start sites, primers and the like may be used, so long as 
the vector constructed provides a cleavage site upstream from and no more 
than one base away from the transcription initiation site. 
The in vitro transcription method provided herein allows for the production 
of functional RNA transcripts, particularly infectious viral RNA 
sequences. The transcription plasmids comprising the desired RNA sequences 
are preferably linearized, preferably with a restriction enzyme 
immediately downstream from the sequences corresponding to the RNA to be 
transcribed. The plasmids are placed in an environment containing an RNA 
polymerase capable of recognizing the promoter sequences. Capping 
sequences, e.g. m7GppG, are also preferably provided when the product 
desired is an infectious viral RNA. Other reactants are provided as known 
to the art, and the reaction mixture is preferably incubated at elevated 
temperatures, preferably about 37.degree. C., until a number of RNA 
transcripts from each plasmid have been generated. These transcripts are 
separated from the reaction mixture and may be used for the purposes 
described above. 
In vitro transcripts of infective RNA's are used in preference to virion 
RNAs because of their freedom cross-contamination and their greater 
genetic definition. 
Because of variation in the properties of commercial preparations of E. 
coli polymerase, it is sometimes difficult to obtain consistent production 
of full-length viral transcripts in good yield. For these and other 
reasons it is desirable to use transcription vectors with other promoters 
from which infectious in vitro transcripts can be produced using other 
types of polymerases, preferably bacteriophage T7 RNA polymerase. This 
phage polymerase has a much higher activity than the bacterial enzyme and 
can be easily purified in high yield from overproducing transformed 
bacterial strains (Davanloo et al. (1984) Proc. Natl. Acad. Sci. USA 
81:2035; Tabor and Richardson (1985) Proc. Natl. Acad. Sci. USA 82:1074). 
One T7-promoted BMV transcript system which has been used in this invention 
involves three plasmids,each containing a cDNA insert corresponding to BMV 
RNA1, 2 or 3, fused in an oligonucleotide-tailored linkage to a T7 
promoter to give transcripts with one additional 5' G preceding the viral 
sequence. This arrangement was selected because it minimizes sequence 
deviations between the first six bases of the resulting BMV transcripts 
and a consensus defined by natural T7 promoters (Dunn and Studier (1983) 
J. Mol. Biol. 166:477). Sequence alterations in this region can adversely 
affect promoter activity (O. Uhlenbeck, personal communication). The 
resulting T7/BMV transcripts are produced in much greater yield per 
plasmid template and have about twice the specific infectivity of previous 
E. coli/BMV transcripts. Much of the infectivity increase may be due to a 
greater proportion of full-length product RNA synthesized by the more 
transcriptionally active and RNAse-free T7 polymerase. 
Addition of a single extra 5' G residue has no significant effect on the 
infectivity of transcripts from cloned BMV cDNA, but addition of 7- or 
16-base 5' extensions to RNA3 transcripts dramatically suppresses their 
biological activity. Moreover, despite the resultant alteration of the 
promoter consensus sequence, fusion of BMV cDNA directly to the start site 
of the canonical .phi.10 promoter of bacteriophage T7 allows efficient in 
vitro synthesis of infectious BMV transcripts by T7 RNA polymerase, 
providing a substantial improvement in the ease and reproducibility with 
which BMV cDNA can be expressed. Uncapped BMV transcripts were found to be 
infectious to barley protoplasts, although at a lower efficiency than 
capped transcripts. 
Structural features at or near the 5' and 3' ends of many viral RNAs are 
thought to have important roles in viral replication and expression. Not 
surprisingly, then, structural alterations at the 5' end of BMV 
transcripts can significantly modulate infectivity. In particular, the 
presence of a 5' cap markedly enhances infectivity, while the presence of 
additional nonviral 5' bases can significantly reduce infectivity. 
A variety of independent results demonstrate that presence of a 5' cap 
structure considerably enhances stability and translational messenger 
activity of many RNAs in eukaryotic cells (Shih et al. (1976) J. Virol. 
19:736; Shimotohno et al. (1977) Proc. Natl. Acad. Sci. USA 74:2734; 
Contreras et al. (1982) Nucleic Acids Res. 10:6353; Green et al.(1983) 
Cell 32:681). BMV virion RNA is naturally capped, and in vitro transcripts 
from either E. coli or phage polymerases can be efficiently and 
conveniently capped by a simple nucleotide substitution method (Contreras 
et al. (1982) supra; Konarska et al. (1984) Cell 38:731). Such capping 
considerably enhances infectivity of BMV in vitro transcripts (Ahlquist et 
al. (1984) Proc. Natl. Acad. Sci. USA 81:7066). In initial whole plant 
infectivity studies, capped BMV transcripts were readily infectious to 
whole plants, but uncapped transcripts were not detectably infectious. In 
protoplasts, where a vastly higher percentage of cells are primarily 
infectable, uncapped transcripts are detectably infectious, but with an 
efficiency reduced at least 10-fold compared to capped transcripts. 
Protoplasts inoculated with all three BMV transcripts, of which two are 
capped and one uncapped, show that viral RNA replication is highly 
inhibited by failure to cap either RNA 1 or 2, but is little affected by 
failure to cap RNA3. That is, in protoplasts co-inoculated with capped BMV 
1 or 2 transcripts, uncapped BMV3 transcripts can be nearly as effective 
as capped transcripts in inducing synthesis of progeny RNA3 (R. French, 
unpublished results). This differential capping sensitivity may result 
from the need for translation products from both RNA 1 and 2, but not 
RNA3, to initiate viral RNA replication (Kiberstis et al. (1981) virology 
112:804; French et al. (1986) Science 231:1294). Rapid initiation of 
replication, through enhanced expression of capped BMV 1 and 2 
transcripts, overcomes reduced stability of uncapped BMV3 transcripts by 
producing new and more stable viral RNA copies in first (-) and then (+) 
strand form. In all the protoplast experiments, a BMV transcript capped 
with a natural methylated 5' end (M7GppG . . .) was about twice as active 
as a transcript with an unmethylated cap (GppG . . .). 
Although the T7-promoted BMV transcripts described above are highly 
infectious while bearing an additional nonviral 5' G, larger nonviral 5' 
extensions can seriously decrease the infectivity of the transcript. The 
5' structures of four complete BMV3 in vitro transcripts whose infectivity 
was examined had either 16, 7, 1 or no additional nonviral bases at the 5' 
end. When inoculated onto protoplasts with BMV 1 and 2 transcripts with 
natural 5' ends, each of these transcripts produced progeny RNA3. However, 
the yield of progeny RNA3 from BMV3 transcripts with 5' extensions of 7 
and 16 bases was 20- to 100-fold less than the yield from transcripts with 
one or no extra 5' bases. 
In contrast with the 5' end results, a transcript with 6-7 additional 
nonviral 3' bases can have nearly the same infectivity as one with a more 
natural 3' end. We have constructed and tested two different 3' ends on 
BMV transcripts, both generated by polymerase runoff at a DNA restriction 
cleavage site. One 3' end is generated by runoff transcription at a PstI 
site fused directly to the 3' end of BMV cDNA. complete transcription of 
the template DNA strand at this site would yield a product RNA lacking the 
terminal A residue of mature BMV virion RNA. However, because of the 
tRNA-like properties of this end, such an incomplete RNA can be processed 
to mature form by host cell ATP, CTP: tRNA nucleotidyl transferase (Hoshi 
et al. (1983) EMBO J. 2:1123). Since this transferase can add all three of 
the terminal -CCA(3'OH) residues to tRNA, repair would also be expected if 
transcription terminated one or two residues upstream of the PstI DNA end. 
Available evidence also suggests that the normal pathway of BMV virion RNA 
synthesis involves similar non-template-directed addition of the terminal 
A in consequence of (-) strand initiation at the penultimate residue of 
the (+) strand template (Miller et al. (1986) J. Mol. Biol. 187:537; 
Collmer and Kaper (1985) Virology 145:249). Thus the "PstI" 3' transcript 
end may duplicate a natural BMV replication intermediate, and it is not 
surprising that transcripts with this 3' end show a high level of 
infectivity. Another 3' end tested with BMV transcripts is generated by 
runoff termination at an EcoRI site shortly downstream of the BMV cDNA. 
Direct sequencing of such "EcoRI" BMV transcripts shows that about half of 
the transcripts terminate after copying the complete template DNA, while 
the other half terminate one residue before the end of the DNA (Ahlquist 
and Janda (1984) Mol. Cell. Biol. 4:2876). This gives rise to a population 
of transcripts with either six or seven additional 3' bases. The 
infectivity of these 3'-extended EcoRI transcripts has not been 
distinguishable from that of the more natural PstI transcripts. Since the 
additional bases on the EcoRI end must block 3' aminocylation of the 
transcript, this would indicate that aminocylation of BMV RNA is not 
required in the initial stages of infection. 
The following examples are provided by way of illustration and not by way 
of limitation. 
EXAMPLE 1 
Construction and Screening of Phage Recombinants M13/PR1-9 
Plasmid pCQV2 (Queen (1983) J. Mol. Appl. Genet. 2:1-10) was cleaved with 
restriction endonucleases ClaI and BamHI and the fragment containing the 
phage lambda cI.sup.ts -P.sub.RM -P.sub.R -ATG sequences was isolated from 
a low-melting point agarose gel (Sanger et al. (1980) J. Mol. Biol. 
143:161-178) and ligated into calf intestinal phosphatased AccI+BamHI cut 
M13mp9 RF DNA using T4 DNA ligase. This DNA was used to transform 
competent E. coli JM101 cells and plated. Nine white plaques were selected 
and screened by the ddATP sequencing technique (Sanger et al. (1982) JMB 
162:729-773). The resulting autoradiograph confirmed that eight of the 
nine phage clones contained the expected phage lambda DNA insert, the 
remaining clone being a deletion mutant of M13mp9. One of the M13/.lambda. 
recombinants, designated M13/PR1, was selected for further work. 
EXAMPLE 2 
Optimum Primer Concentrations for Mismatch Priming on M13mp9/pPR1 ssDNA 
The 15-mer mismatched oligonucleotide 5'-OH-d(GGGACCATTATCACC)-3'-OH which 
will be referred to as either the P.sub.R -oligonucleotide or the P.sub.R 
primer, was synthesized by the phosphite triester method. This P.sub.R 
primer was designed in such a manner as to allow a precise alteration of 
the phage lambda P.sub.R promoter. A number of ways were used to test the 
priming of this P.sub.R primer on P.sub.R DNA. Because of the presence of 
the complementary stem and loop in the nucleotide sequence of interest 
(see supra), the following experiments were designed to test whether 
complete denaturation of the M13mp9/pPR1 DNA template plus vast excesses 
of the P.sub.R -primer could achieve priming at the lambda P.sub.R 
promoter and whether such primers annealed at this site could be extended 
by reverse transcriptase without extension of P.sub.R primers bound at the 
upstream cI.sup.ts site. It will be noted (FIG. 5) that when the P.sub.R 
primer is bound at the upstream cI.sup.ts site that it cannot then pair at 
the extreme 3' terminus. Such a lack of pairing at the extreme 3' terminus 
should prevent extension by reverse transcriptase which lacks a 
3'-exonuclease activity. Therefore, M13PR1 ssDNA was mixed with P.sub.R 
oligomer at 1, 10, 100, and 1000-fold molar excesses, annealed by the 
boiling/slow cooling method (Anderson et al. (1980) Nucleic Acids Res. 
8:1731ff) and used as a template/primer complex in ddTTP sequencing 
reactions using reverse transcriptions (Ahlquist et al. (1981) Cell 
23:183-189). Autoradiography of the final gel showed that under these 
conditions DNA synthesis was primed exclusively from the desired site 
within the P.sub.R promoter sequence. Efficiency of DNA synthesis 
increased steadily with P.sub.R primer concentration, being at the highest 
primer:template ratio (1000:1) as efficient as synthesis from a 5-fold 
excess of the normal M13/lac sequencing primer (Duckworth et al., Nucl. 
Acids. Res. 9:1691-1706). 
EXAMPLE 3 
Use of the PR Oligonucleotide to synthesize and Clone an Altered Promoter 
Sequence 
A 1000-fold molar excess of P.sub.R oligomer was mixed with approximately 
2.mu.g M13/PR1 ssDNA and annealed by the boiling/slow cooling method 
(Anderson et al. (1980) Nucl. Acids Res. 8:1731ff). The resulting 
template/primer complexes were extended by reverse transcriptase in the 
presence of all four dNTPs. The product DNA was isolated by ethanol 
precipitation and used in a second DNA synthesis reaction primed with an 
M13 lac "reverse" primer as described by Hong (Bioscience Reports 
1:243-252 (1981)). The resulting DNA products were cleaved with PstI, 
electrophoresed on a low-melting point agarose gel, and the 800-1000 bp 
fraction, as assayed by the mobility of defined marker DNA fragments, 
excised and eluted (Sanger et al. (1980), J. Mol. Biol. 143:161-178). This 
DNA was then mixed with SmaI+PstI cut M13mp9 RF DNA. treated with T4 DNA 
ligase, and used to transform competent E. coli JM101 cells. After 
plating, 30 white plaques were selected and screened by the ddATP 
sequencing method (Sanger et al. (1982) JMB 162:729-773). The pattern 
obtained for 16 of these recombinants was consistent with the desired 
construct in which the sequence of the lambda P.sub.R promoter element was 
linked directly to the vector SmaI site. 
Since the purpose of the PM clone constructions was to obtain a particular 
altered promoter sequence (i.e., a SmaI restriction endonuclease site), 
two of these recombinants, designated M13 PM86 and M13 PM87, were 
sequenced by the 2', 3'-dideoxynucleoside triphosphate method (Sanger, F. 
et al. (1977) Proc. Nat. Acad. Sci. U.S.A. 74:5463-5467). The nucleotide 
sequence in the vicinity of the altered P.sub.R promoter in both strains 
was exactly as desired, namely: 
##STR2## 
The nucleotide sequence changes that have been made at the SmaI site can be 
seen by a comparison of the above sequence with that of PB3PR13 (FIG. 2). 
The sequence of M13 PM86 was read for a further 210 bases upstream of the 
sequence shown above. It was then computer matched to the sequence of 
pCQV2 (Queen, C. (1983) J. Mol. Appl. Genet. 2:1-10) without any 
discrepancies. Close visual examination of the autoradiograph revealed no 
sequence differences between M13 PM86 and M13 PM87. 
EXAMPLE 4 
Subcloning M13/PM86,87 Inserts into pUC9 
For each of the two clones M13 PM86 and M13 PM87, RF DNA was digested with 
the restriction endonucleases PstI and EcoRI and the EcoRI-PstI fragment 
containing the desired modified P.sub.R promoter was recovered following 
electrophoresis in low melting point agarose. The appropriate DNA band (as 
judged by size) was excised, the DNA extracted as previously described 
(supra), and ligated to EcoRI+PstI cut pUC9 (Vieira and Messing (1982) 
Gene 19:259-268). This DNA was used to transform competent E. coli MJ101 
cells which were plated on media containing ampicillin, X-gal and IPTG. 
EcoRI and EcoRI+PstI digests of minilysate DNA samples (Ish-Horowicz and 
Burke (1981) Nucl. Acids. Res. 9:2989ff) from six white colonies from each 
ligation mix were examined by agarose gel electrophoresis. In each case 
the digests confirmed the expected size of the insert fragment and that 
only one such fragment had been inserted. One of these clones, an M13/PM86 
derivative designated pPM1, was selected for further work. Transcripts of 
pPM1 were obtained by standard methods. Runoff reverse transcription of 
these transcripts showed that initiation on the P.sub.R promoter occurred 
precisely at the SmaI site as planned (illustrated below). 
##STR3## 
EXAMPLE 5 
Insertion of a Complete cDNA Copy of Brome Mosaic Virus RNA3 (BMV RNA3) 
Into the SmaI Restriction Endonuclease Cleavage Site of pPM1 
The nucleotide sequence of BMV RNA3 is known (Ahlquist, P. et al. (1981) J. 
Mol. Biol. 153:23-28). Starting from this information, synthetic 
oligonucleotide primers for the first and second strands of BMV RNA3 cDNA 
were designed, synthesized and used to prime production of ds cDNA to BMV 
RNA3 (Fields and Winter (1982) Cell 28:303-313). Full lengths ds cDNA was 
isolated from an agarose gel (supra) and inserted into the SmaI site of 
pPM1. Clones in which the cDNA was oriented to give (+) strand transcripts 
(i.e., transcripts corresponding in polarity to encapsidated viral RNA) 
were selected after screening by restriction digestion. Plasmid DNA from 
such selected clones was cut with EcoRI and transcribed in vitro by 
standard methods. Results obtained using runoff reverse transcription of 
the transcripts showed that transcription initiation occurred on the first 
nucleotide downstream from the SmaI cleavage site of the pPM1 
transcription vector. 
EXAMPLE 6 
Synthesis of infectious BMV transcripts with bacteriophage RNA polymerases 
Using standard techniques including restriction endonuclease digestion, DNA 
ligation, synthetic oligonucleotide-directed deletion, bacterial 
transformation and DNA sequencing (Maniatis et al. (1982) Molecular 
Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; 
Biggin et al. (1983) Proc. Natl. Acad. Sci. USA 80:3963-3965), fusions 
were made between bacteriophage T7 and SP6 promoters and each of the BMV 
cDNA inserts from clones pB1PM18, pB2PM25 and pB3PM1 (Ahlquist and Janda 
(1984) Mol. Cell. Biol. 4:2876-2882). These plasmids were deposited with 
the American Type culture Collection, Rockville, Md. on March 7, 1984 as 
Nos. 40171 and 40169 respectively. 
The cDNA insert of a biologically active BMV RNA3 clone was inserted in the 
transcription vector pGEM1 available from Promega. The resultant plasmid, 
pB3GE1, allowed SP6 RNA polymerase transcription (Melton et al. (1984) 
Nucleic Acids Res. 12:7035-7056) of an RNA containing the entire BMV RNA3 
sequence plus 6 extra nonviral nucleotides at its 5' end (Table 1). In 
initial barley protoplast tests, this transcript was around 100 times less 
infectious than pB3.sub.PM 1 transcripts, showing that additional nonviral 
5' nucleotides could reduce BMV transcript infectivity. 
RNA3 cDNA was then fused in closer proximity to bacteriophage T7 late 
promoter .phi.10 (Dunn and Studier (1983) J. Mol. Biol. 166:477-535) 
(provided by John Dunn in the form of plasmid pAR2463). The fusions were 
constructed between the PstI and EcoRI sites of plasmid pUC119. This 
plasmid is available from Jeffrey Vieira of the Waksman Institute, Rutgers 
University, and reproducibly described in Vieira and Messing (1987) Meth. 
Enzymol. 153:3ff. Because the consensus sequence of natural T7 late 
promoters extends 6 bases into the transcript, and the effects of sequence 
changes in this region on promoter activity were not known, two different 
oligonucleotide-directed fusions were made (Table 1). After linearization 
with EcoRI, plasmids were transcribed in reactions containing 20 ng/.mu.l 
DNA, 0.2 .mu./.mu.l T7 RNA polymerase (Promega Biotec), 400 .mu.M rATP, 
rCTP, rTTP, 75 .mu.M rGTP, 500 .mu.m m7GpppG or GpppG, 40 mM Tris-Cl pH 
7.9, 10 mM NaCl, 6mM MgCl.sub.2, 10mM DTT, 2mM spermidine and 1 
.mu./.mu.l placental ribonuclease inhibitor (Promega Biotec) for 60 min. 
at 37.degree. C., producing typically 10-20 RNA transcripts per plasmid 
template. After transcription, RNAse-free DNAse I (Promega Biotec) was 
added to 1 .mu./.mu.l and incubated 15 min. at 37.degree. C. The reaction 
was extracted once with 1:1 phenol-chloroform, NaAc added to 0.3M, RNA 
precipitated with ethanol, ethanol washed, dried and dissolved in a small 
volume of ddH.sub.2.sbsb.0. This produced transcripts with either one 
extra 5' G residue (pB3TP7) or no extra 5' residues (pB3TP8). The first 
fusion was chosen to minimize changes in the promoter consensus region, 
while the second was chosen because of the proven infectivity of 
correctly-initiated BMV transcripts. A third fusion of RNA3 cDNA to the T7 
promoter, including seven extra 5' nucleotides in the transcript (pB3TP5), 
was obtained as an intermediate in the construction of the two closer 
fusions (Table 1). Despite the sequence alterations at the transcription 
start site, all three T7/BMV3 plasmids gave equal yields of around 10-20 
copies per plasmid template when transcripts confirmed that initiation 
occurred at the positions predicted in Table 1. Protoplast infectivity 
tests with B1.sub.PM and B2.sub.PM transcripts showed that pB3TP7 and 
pB3TP8 transcripts had a much higher infectivity than pB3TP5 transcripts. 
Based on these results, oligonucleotide-directed fusions of the T7 
promoter to RNA1 and 2 cDNAs were similarly made with either one G residue 
(pB1TP1, pB2TP3) or no residues (pB1TP3, pB2TP5) between the promoter 
initiation site and the cDNA. The same biologically active set of BMV cDNA 
inserts, designated the M1 set was used in all clones in this study. For 
convenience, transcripts produced with bacteriophage T7 or SP6 RNA 
polymerase will be referred to below by the notation Bx.sub.y, where x is 
the BMV component number and y the number of additional nonviral 
nucleotides present in the transcript (Table 1). 
TABLE 1 
__________________________________________________________________________ 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
T7 promoter consensus TAATACGACTCACTAT*GGGAGA 
__________________________________________________________________________ 
DNA sequences at the promotercDNA junctions of several BMV RNA4 cDNA 
clones. The cDNA corresponding to the 5' end of BMV RNA3 (underlined) is 
linked to an SP6 promoter in pB3GE1 and to a T7 promoter in the other 
plasmids. Transcription start sites are shown by a bent arrow, and the 
text designation for each transcript is shown in parentheses. pB3GE1 and 
pB3TP5 were each constructed by sequential subcloning and rejoining of 
suitable restriction fragmdnts from pB3PM1. pB3TP7 and pB3TP8 were derive 
from pB3TP5 by a process involving oligonucleotidedirected mutagenesis of 
suitable M13 subclones. 
EXAMPLE 7 
The infectivities of selected BMV transcript combinations were tested in 
barley seedling inoculations (Table 2). No significant difference in 
infectivity was visible between T7 transcripts with natural 5' ends 
(Bx.sub.0) and those with an additional 5' G (Bx.sub.1) (Experiment 1). In 
combination with B1.sub.0 and B2.sub.0, B3.sub.1 transcripts appeared to 
have a small advantage over B3.sub.0 transcripts (Experiment 2), but when 
the standard deviation between replications of the experiment is 
considered, this difference is not statistically significant (54.+-.18 vs. 
44.+-.16). Additional observations of protoplast infectivity confirmed 
that the activity of the Bx.sub.0 transcripts is not less than the 
Bx.sub.1 transcripts. The 5' ends of virion RNA3 from infections with 
either class of transcript were sequenced and found to be identical to 
that of wild type virus, showing that the additional 5' G of the B3.sub.1 
transcript did not persist in its progeny. Notably, Bx.sub.0 and Bx.sub. 1 
transcripts had a roughly 20-fold infectivity advantage over Bx.sub.PM 
transcripts. This substantial difference is due to the higher transcript 
initiation frequency of T7 RNA polymerase, its greater resistance to 
premature termination under the low rGTP concentrations used for capping 
and a nonlinear BMV infectivity curve due to the need for co-infection by 
all three BMV RNAs. In earlier experiments, achieving 30% infectivity with 
Bx.sub.PM transcripts required the product RNA from around 150 ng of each 
plasmid per plant, compared to the 20 ng used in Table 2 experiments. 
Moreover, the E. coli RNA polymerase used in these earlier experiments 
produced a much higher transcript yield (2-3 copies/plasmid) than 
currently available E. coli RNA polymerase lots from several sources (0.3 
copies or less). This reduced polymerase performance severely affected 
experiments even when E. coli polymerase transcription reactions were 
correspondingly scaled up, and was a major incentive for development of 
the T7 promoter fusions described here. 
The infectivity of RNA3 transcripts with various 5' extensions was compared 
in co-inoculations with B1.sub.0 and B2.sub.0 transcripts (Table 2). In 
contrast to the high activity of B3.sub.0 and B3.sub.1 transcripts, no 
infections were ever seen when B3.sub.7 transcripts were used in the 
inoculum. A few symptomatic plants were seen in experiments 2 of Table 2 
after inoculation with B3.sub.16 transcripts, but this result could not be 
reproduced in multiple later attempts, even with increased inoculum 
concentrations. Infectivity of various BMV transcripts in barley 
protoplasts paralleled the results of whole plant inoculations. BMV RNA 
replication induced by Bx.sub.1 transcripts was similar to that induced by 
Bx.sub.0 transcripts, and was typically manyfold greater than the 
biological activity of Bx.sub.PM transcripts from a similar amount of 
plasmid. Transcripts B3.sub.7 and B3.sub.16 failed to induce RNA3 
replication detectable by direct protoplast labelling. In these cases low 
level replication of RNA3, approaching a few percent of that induced by 
transcripts of pB3TP7 and 8, could be detected by hybridization of 
electrophoretically separated and blotted protoplast RNA to a suitable 
RNA3 probe. Uncapped BMV transcripts were previously not detectably 
infectious to whole plants, and all transcripts in the above experiments 
were capped by inclusion of m7GpppG in the transcription reaction. 
However, uncapped transcripts were detectably infectious to protoplasts, 
although at a lower efficiency than capped transcripts. 
All T7 transcripts in this study were made from plasmid templates 
linearized at an EcoRI site seven bases 3' to the end of the BMV cDNA. In 
addition, we find that T7 RNA polymerase produces infectious transcripts 
with similar efficiency from cDNA templates linearized at a PstI site 
fused to the cDNA 3' end. 
TABLE 2 
______________________________________ 
Whole Plant Infectivity Tests 
Infected plants/ 
Inoculated plants 
Average percent 
Experiment 1 
A B infected 
______________________________________ 
Mock inoculated 
0/21 0/22 0 
BMV virion RNA 
26/45 31/57 56 
B1.sub.0, B2.sub.0, B3.sub.0 
10/42 10/58 20 
B1.sub.1, B2.sub.1, B3.sub.1 
5/42 16/51 23 
B1.sub.PM, B2.sub.PM, 
P3.sub.PM 0/46 1/60 1 
______________________________________ 
Experiment 2 
A B 
______________________________________ 
Mock inoculated 
0/14 0/24 0 
BMV virion RNA 
18/29 33/44 70 
B1.sub.0, B2.sub.0, B3.sub.0 
9/29 23/43 44 
B1.sub.0, B2.sub.0, B3.sub.1 
12/30 26/40 54 
B1.sub.0, B2.sub.0, B3.sub.7 
0/28 0/38 0 
B1.sub.0, B2.sub.0, B3.sub.16 
1/27 4/44 7 
______________________________________ 
7-day old barley plants were inoculated as indicated with no RNA mock), 
virion RNA (50 ng/plant), or in vitro transcripts and scored for the 
presence of mosaic symptoms 7-14 days after inoculation. For each 
experiment, complete independent replications (A, B) were performed on 
separate days. Transcripts were produced from EcoRIlinearized plasmids 
with either T7 or SP6 RNA polymerases (N.E. Biolabs or Promega), except 
that rGTP concentration was reduced to 75 .mu.M and 500 .mu.M m7GpppG 
(N.E. Biolabs) was added. Where used, transcripts from approximately 20 n 
of each plasmid were applied per plant. 
EXAMPLE 8 
To determine which portions of RNA3 contribute to its productive 
replication in infected cells, infectious in vitro transcripts from cloned 
BMV cDNA were used to generate and analyze systematic deletions in RNA3 
cDNA. Suitable deletions, bounded either by specific restriction sites or 
Bal31 digestion end points, were constructed in plasmid pB3PM1, in which 
RNA3 sequences are fused to an E. coli RNA polymerase promoter or in 
plasmids pB3TP7 and pB3TP8 (having the T7 promoter). In vitro 
transcription of such clones produced deleted versions of RNA3 which were 
tested for their ability to be replicated in barley protoplasts 
coinoculated with RNA1 and 2 cDNA transcripts. Net viral RNA synthesis in 
infected cells was assayed after gel electrophoresis by hybridization with 
an RNA probe complementary to the conserved 3' terminal 200 bases of BMV 
genomic and subgenomic RNA's. Protoplasts inoculated with only RNA1 and 2 
transcripts, or with all three wild type genomic transcripts, served as 
controls. Such hybridization assays allowed detection of RNA3 at levels 
several hundred fold lower than that produced during wild type infection, 
and the extent of RNA1 and RNA2 replication as a convenient internal 
standard to assess the efficiency of each inoculation and the ability of 
each protoplast sample to support viral RNA replication. Amplification of 
each RNA3 variant was quantitated by densitometry of autoradiographs and 
the results expressed as per cent accumulation relative to wild type RNA3 
after normalization for inoculation efficiency as measured by the amount 
of RNA1 produced in the infection. 
Deletions in the 3a gene: Deletions of 100-500 bases were made in either 
direction from a central ClaI site in the 3a gene and tested for progeny 
accumulation in protoplasts. Multiple deletions in the 3a gene were 
tested, showing that the entire gene or any portion thereof can be deleted 
with little or no effect on RNA3 accumulation. Deletions from the central 
ClaI site to within 25 bases of the 3a gene 5' end to the exact 3a gene 3' 
end, or between both extremes all directed progeny RNA accumulation nearly 
as efficiently as wild type RNA3. RNA3 replication thus has no specific 
requirement for either the RNA sequences or protein product of the 3a 
gene. RNA1 and 2 replication is independent of RNA3, and neither RNA3 nor 
RNA4 synthesis requires coat protein. (French, R., et al. (1986) 
"Bacterial gene inserted in an engineered RNA virus: Efficient expression 
in monocotyledonous plant cells," Science 231:1294-1297 and Kiberstis, P. 
A., et al. (1981) "Viral protein synthesis in barley protoplasts 
inoculated with native and fractionated brome mosaic virus RNA," Virology 
112:804-808.) Taken together, these results indicate that synthesis of the 
four major species of BMV RNA does not require known RNA3 gene products. 
Preliminary results from a finer scale deletion mapping of sequences 
extending into the 5' noncoding region show that RNA3 accumulation is 
unaffected until deletions enter the noncoding region itself (bases 1-91), 
with a decline in replicative ability of the mutants as the deletion 
endpoint moves closer to the 5' end. 
Deletions in the coat gene and 3'-noncoding region: Deletion of a major 
segment of the coat gene, extending from the SalI site at the beginning of 
the coat gene to the XbaI site near the end of the gene, has only moderate 
(three-fold) effect on either RNA3 or 4 production during infection. 
(French, R., et al. (1986), supra.) Additional deletions extending further 
in both directions were made and tested in barley cells. Three of these 
have increasing amounts of 3'-noncoding sequence removed, retaining 200, 
162 and 100 bases, respectively, of the 3' terminus. Of these, the one 
with 200 bases retained accumulated to 40 per cent of the wild type level, 
the one with 162 retained accumulated to levels several hundred-fold lower 
than wild type RNA3, and the one with 100 bases retained had undetectable 
progeny. Similar to previously tested deletions within the coat gene, the 
one with 200 bases retained also generated a small subgenomic RNA with 
predicted length of only 216 bases. Extending the 5' boundary of coat gene 
deletions from the SalI site just within the coat gene to the BglII site 
20 bases of 5' of the subgenomic mRNA initiation site had two notable and 
possibly related effects. Presumably because the subgenomic initiation 
site was removed, no detectable subgenomic RNA was produced. In addition, 
genomic RNA3 accumulation for BglII-bounded deletions was several-fold 
above that of the analogous SalI-bounded deletions such as the one with 
200 3'-noncoding bases retained. 
Deletion of the intercistronic region seriously inhibits RNA3 replication: 
Although the results above show that some sequences of the RNA3 
intercistronic region may be deleted without affecting RNA3 production, 
any of a variety of deletions into or across the intercistronic region 
dramatically reduce RNA3 accumulation, by up to several hundred fold. 
Progressive deletions from either side of the intercistronic region were 
used to localize the area responsible for this behavior. As deletions 
extended from 48 to 140 bases into the 5' side of the intercistronic 
region, RNA3 accumulation was progressively reduced. In each case, the 
level of RNA4 was also reduced so that the molar ratio of RNA4:RNA3 
remained near the 1:1 ratio of wild type RNA3. A deletion extending from 
the ClaI site near the center of gene 3a (which extends to about base 
1000), to base 1015 replicated at near wild type levels, indicating that 
the 5' boundary of the active region defined by these assays lies between 
bases 1015 and 1051. The 3' boundary of required intercistronic sequences 
identified above does not extend beyond the BglII site near the beginning 
of the RNA4 gene. This 3' boundary was further mapped using deletions 
extending from within the coat gene to various endpoints 5' to the BglII 
site. Repeated experiments revealed small but progressive reductions in 
RNA3 accumulation until such deletions extend into or beyond the vicinity 
of the intercistronic AvaI site (base 1147). When this occurs, RNA3 
accumulation is again reduced several hundred fold. Consequently, the 
intercistronic region with maximal effect on RNA3 accumulation does not 
include the internal oligo(rA) sequence of heterogenous length which 
occurs immediately 5' to the BglII site. 
While these results indicate that a subset of the intercistronic region 
contributes to RNA3 accumulation, all the intercistronic deletions had at 
least one end point well inside adjoining coding regions. To confirm that 
the effect was entirely due to deletion of intercistronic sequences, three 
additional mutants with smaller deletions were constructed. Two of these 
had only intercistronic sequences deleted (bases 1013-1142, and bases 
1013-1221), and a third (bases 1013-1253) deleted only to the beginning of 
the coat cistron. Even this small deletion in the coat cistron region 
reduced the level of RNA3 to nearly the same extent as the larger 
deletions, confirming that the intercistronic sequences upstream of the 
oligo(rA) are important for RNA3 accumulation. In addition, a 221 base 
intercistronic segment encompassing the entire region implicated above 
(bases 1004-1225) was inverted in the strongly replicating variant 
containing bases 604-1225. Similar to deletion of the entire 
intercistronic region, accumulation of not only (+) but also (-) strand 
RNA was severely inhibited with the reversed variant, showing that this 
intercistronic segment functions only in appropriate orientation relative 
to other elements of BMV RNA3. 
Simultaneous deletions in the 3a and coat genes: As noted above, nearly 
complete or complete deletion of either the 3a or coat genes had only 
small effects on production of RNA's 3 or 4. However, when major deletions 
in both coding regions occurred simultaneously in the same molecule, 
(bases 101-1003 and 1258-1760; bases 117-601 and 1258-1917; and bases 
117-1003 and 1258-1917), RNA3 levels were again reduced over one hundred 
fold. Among tested mutants with deletions in both genes, only one with 
deletion of bases 604-1003 and 1258-1917 which retained the 5' half of the 
3a gene and has the least total amount of sequence removed, accumulated to 
readily visible amounts, but was still depressed compared to wild type 
RNA3. A possible, but unlikely explanation for these results was that 
expression of either the coat protein or the N-terminal domain of the 3a 
protein, though not both, was required for efficient RNA 3 amplification. 
To test this, an RNA3 derivative with deletions of bases 100-110 and 
1253-1254 with frameshift mutations just within the 5' boundaries of both 
the 3a and coat cistrons was constructed. This mutant both replicated and 
generated subgenomic RNA at levels comparable to wild type RNA3, 
confirming that neither RNA3 nor RNA4 synthesis depends on the known RNA3 
gene products. 
The mutant with deletions from bases 101-1003 and 1258-1760 was further 
modified by inserting the bacterial chloramphenicol acetyltransferase 
(CAT) gene sequence in place of the deleted coat gene. Despite its 
heterologous nature, this insertion dramatically improved RNA3 
accumulation, giving 17 per cent of the wild type RNA3 level. This equals 
the accumulation of an RNA3 derivative with an intact 3a gene and the same 
CAT insertion, suggesting that the remaining reduction of accumulation 
relative to wild type RNA3 is due to the presence of CAT sequences rather 
than the absence of BMV sequences. 
Effects of deletions and rearrangements on accumulation of RNA3 (-) 
strands: Normal BMV infections induce highly asymmetric production of (+) 
and (-) viral RNA strands, with wild type RNA3 yielding about a 100-fold 
excess of (+) strands after 20 hour infection of barley protoplasts. The 
relative levels of (-) and (+) strand genomic RNA accumulation were also 
tested for a number of deletion mutants discussed above. These levels were 
tested by probing parallel blots of the same gel-electrophoresed RNA 
samples with suitable (+) and (-) strand probes. Reactivity and specific 
activity of the two different probes was normalized by comparison of the 
hybridization signals of BMV RNA standards of known concentration. For all 
variants tested, the ratio of (+) to (-) strand RNA3 was similar to that 
of wild type BMV, indicating no significant differential in overall 
effects on RNA3 (+) or (-) strand accumulation. 
The foregoing examples are illustrative of this invention, and as will be 
understood by those skilled in the art, many equivalent constructions and 
organisms may be prepared by methods analogous, similar or otherwise 
equivalent to those, specifically discussed herein. For example, because 
transcripts having one extra 5' base have been found to be functionally 
equivalent to those having no extra 5' bases, vectors functionally 
equivalent to pPM1 and other similar vectors allowing insertion of a 
nucleotide sequence to be transcribed at a restriction site whose cleavage 
site is immediately upstream from the transcription initiation nucleotide 
are those in which the transcription initiation nucleotide is immediately 
upstream of the cleavage site, so that there is one additional base on the 
5' end of the transcribed sequence. Similarly, vectors equivalent to pPM1 
with nucleotide sequences inserted therein which produce transcripts with 
no additional 5' bases are similar vectors having nucleotide sequences 
inserted therein which produce transcripts having one additional 5' base. 
Plasmid pPM1 was deposited Mar. 7, 1985, with the American Type Culture 
Collection, 12301 Parklawn Drive, Rockville, Md., as No. 40172.