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The present invention relates to a picornaviral genetic sequence that is
capable of improving the efficiency of translation of RNA into
proteinaceous material. More particularly, it relates to a 5' non-coding
cardiovirus nucleotide sequence capable of enhancing such translation in
cell free media.
BACKGROUND OF THE INVENTION
With the increasing use of genetic engineering techniques, commercial scale
production of many proteinaceous materials has become possible. Once a
protein becomes available in large quantity, researchers can then more
easily study it and its function, and they can use it for other purposes
that require relatively large quantities of the protein. Of course, even
where commercial scale production techniques are available, there is
always a desire to lower the cost and time involved in the production of
such proteins.
One technique that the art has tried to make production more efficient is
to locate efficient DNA promoter sequences that occur in nature, and then
use them in order to improve the efficiency of "transcription" of DNA to
RNA. Another approach has been to develop means to culture eukaryotic host
cells, so as to provide suitable hosts to permit "translation" (RNA to
protein) of certain proteins that need host proteins to be expressed. This
approach is costly, involves separation problems, and is not universally
applicable.
Yet another approach is to create "cell free" extracts (e.g. derived from
rabbit reticulocytes) so as to provide an in vitro media for translation
of RNA to proteinaceous material. See e.g. D. Shih et al., 30 J. Virol.
472-480 (1979); S. Gupta et al., 144 Virol. 523-528 (1985). The disclosure
of these articles (and all of the other articles recited herein) are
incorporated by reference as if fully set forth below. Unfortunately, in
vitro translation of certain RNA (e.g. polio virus and rhinovirus) has
been inefficient in such media.
In the past, researchers have conducted general research into various
viruses known as cardioviruses. These picornavirus RNA viruses do not go
through a DNA stage in their life cycle. Encephalomyocarditis virus
("EMC"), Mengovirus, Mous-Elberfeld virus, MM virus, and Columbia SK virus
are examples of cardioviruses. It has been learned that these viruses have
relatively efficient translation of their coding RNA in cell free systems.
It has also been learned that there are various techniques for culturing
and producing the natural cardioviruses. Some work has also been done on
sequencing these viruses. See generally H. Pelham, 85 Eur. J. Biochem.
457-463 (1978); A. Palmenberg et al., 32 J. Virol. 770-778 (1979); A.
Palmenberg, 41 J. Virol. 244-249 (1982); R. Rueckert et al., 78 Meth.
Enzym. 315-325 (1981); C. Shih et al., 40 J. Virol. 942-945 (1981); A.
Palmenberg, 44 J. Virol. 900-906 (1982); A. Palmenberg et al., 12 Nucl.
Acid. Res. 2969-2985 (1984); R. Rueckert et al., 50 J. Virol. 957-959
(1984); K. Chumakov et al., 246 Dokl Biochem 209-212 (1979).
SUMMARY OF THE INVENTION
In one aspect of the invention there is provided a DNA sequence coding for
an RNA translational enhancer. The enhancer has the characteristics of a
cardiovirus RNA translational enhancer sequence that is located 5' of a
cardiovirus AUG sequence. Preferably, the RNA translational enhancer is of
the non-coding type, the cardiovirus is encephalomyocarditis, and the DNA
sequence has the characteristics of an encephalomyocarditis translational
enhancer coding region in ATCC 67525. A host, e.g. E. coli, can contain
the DNA sequence.
In another aspect of the invention, there is provided a recombinant DNA
vector. The vector has a DNA transcriptional promoter and a DNA enhancer
coding sequence capable of coding for an RNA translational enhancer. The
enhancer has the characteristics of a cardiovirus RNA enhancer sequence
that is located 5' of a cardiovirus AUG sequence. The DNA enhancer coding
sequence is positioned on the vector so as to be subject to the
transcriptional promoter. There is also a foreign DNA gene (e.g. one
capable of producing a foreign proteinaceous material of interest) which
is positioned on the vector so as to be subject to the transcriptional
promoter and so that after transcription of the foreign DNA gene and the
DNA enhancer coding sequence to their RNA variants, translation of the RNA
variant of the foreign DNA gene will be subject to the control of the RNA
variant of the enhancer coding sequence.
In a preferred form, the RNA translational enhancer is of the non-coding
type, the cardiovirus is encephalomyocarditis, the foreign DNA gene does
not code for a cardiocvirus protein, and the enhancer coding sequence has
the characteristics of an encephalomyocarditis translational enhancer
coding region in ATCC 67525.
In yet another aspect of the invention, a recombinant RNA sequence is
provided. It has an RNA translational enhancer of the noncoding type which
in turn has the characteristics of a cardiovirus RNA translational
enhancer sequence that is located 5' of a cardiovirus AUG sequence (and
preferably 3' of a poly C tract). There is also provided a foreign RNA
sequence linked to the enhancer so as to be subject to the control of the
enhancer.
In another embodiment of the invention, there is provided a method of
producing a desired proteinaceous material. According to the method, one
expresses, in vitro, in a cell free media, the above foreign RNA sequence
using the recombinant RNA sequence.
An object of the invention therefore is to provide DNA and RNA sequences,
vectors, and hosts of the above kind which can be used to improve RNA
translation efficiency.
Another object of the invention is to provide DNA and RNA sequences,
vectors, and hosts of the above kind, together with methods for their use,
to enable a desired proteinaceous material to be produced in vitro, in a
cell free media, without extraneous enhancer coding sequences being
attached to the proteinaceous material which is produced.
Another object of the invention is to provide an improved means of
expressing virion proteins of picornaviruses.
Still other objects and advantages of the present invention will be
apparent from the description which follows.
DESCRIPTION OF THE FIGURES
FIG. 1 shows a schematic depiction of the encephalomyocarditis RNA genome;
FIG. 2 shows a schematic depiction of a DNA plasmid incorporating the DNA
variant of an enhancer of the present invention; and
FIG. 3 shows the RNA nucleotide sequence of nucleotides 1-3 and 260-840 of
the EMC genome.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
It should be understood that the descriptions of the preferred embodiments
below are merely examples of the invention. They are not intended to
represent the full scope of the invention. Rather, the claims should be
looked to in order to determine the full scope of the invention.
The RNA genome of EMC is shown in FIG. 1. The first 834 nucleotides of the
almost 8000 EMC RNA nucleotides are noncoding nucleotides located 5' of
the AUG coding initiation site. Over 200 of these 834 nucleotides are
repetitive C's (the poly C tract). The open box portions of FIG. 1
represent the coding portions of the genome. Also shown are the positions
of some restriction sites of interest.
CONSTRUCTION OF A RECOMBINANT DNA VECTOR
The research which led to the present invention is described in somewhat
greater detail than below in G. Parks et al., 60 J. Virol. 376-384 (Oct.
17, 1986) (not prior art). Restriction enzymes were purchased from New
England Biolabs. DNA manipulations were done using standard methods. See
e.g. T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, Laboratory (1982). All transformations to ampicillin
resistance were performed with E. Coli HB101 obtained from Promega Biotec,
Madison, Wis.
Culturing and purification of EMC viral RNA was as described in R. Rueckert
et al., 78 Meth. Enzym. 315-325 (1981). Random length RNA segments were
created with various restriction enzymes. To change viral RNA segments to
double stranded DNA, reverse transcription procedures analogous to those
reported in A. Palmenberg et al., 12 Nuc. Acid Res. 2969 (1984) were
followed. EcoRI DNA linkers (New England Biolabs) were then ligated to the
ends of the double-stranded DNA, and the resulting material was inserted
into the EcoRI site of the transcription vector pSPT18 (Pharmacia Fine
Chemicals). Plasmid pSPT18 is a derivative of plasmid pUC18 containing a
T7 transcriptional promoter sequence and a polylinker cloning site. (See
FIG. 2)
One of the resulting plasmids, plasmid pE3Tll contained EMC sequences
originating from within the poly(C) tract and extending 3' about 2,300
bases past the AUG into the coregion encoding viral peptide VP3. To
construct plasmid pE5LVP0, DNA from plasmid pE3Tll (1 .mu.g) was digested
with XbaI to completion. After extraction with phenol-chloroform and
precipitation with ethanol, the DNA was reacted with T4 DNA ligase to
reform the plasmid, and a portion of the mixture was used to transform E.
coli HB101. The resulting colonies were screened for the size of the EMC
segment. One plasmid was chosen and designated pE5LVP0. It contains DNA
corresponding to nucleotides 260 through 2004 of EMC RNA. pE5LVP0 in E.
coli C600 Amp.sup.R has been deposited with the American Type Culture
Collection, Rockville, Md., with ATCC #67525. It will be made available as
required under applicable patent law. Such availability is not intended as
a license to practice the invention.
As shown in FIG. 3, nucleotides 834-36 are the AUG coding initiation
sequence. AUG in RNA corresponds to ATG in DNA and ATGGCCA on the DNA
includes the BalI site TGGCCA. Thus, after digestion of the plasmid with
XbaI and BalI, a plasmid is derived with no EMC coding regions, and a
foreign gene can be inserted at the cut point, followed by ligation to
recircularize.
An easier alternative is to provide a desired DNA foreign gene with a
terminator sequence, and then insert it at the BalI site of pE5LVP0. For
example, DNA (4 .mu.g) from a full-length clone of polio type I Mahoney
virus (Racaniello, et al., 214 Science 916-919 (1981)) can be digested
with NruI (2 units) for 15 hours at 37.degree. C, followed by digestion
with SmaI (5 units) at 30.degree. C for 12 hours. The resulting fragment
is ligated for 12 hours at 12.degree. C with 10 units of T4 DNA ligase and
0.5 .mu.g of plasmid pE5LVP0, which has previously been digested with 2
units of BalI for 12 hours at 37.degree. C. What is formed by this latter
technique is a recombinant plasmid vector with a transcriptional promoter
T7, followed by a DNA translational enhancer non-coding region, a foreign
DNA gene (in this case the 3' half of the polio type I Mahoney coding
region), a terminator, and then the rest of the plasmid.
To transcribe the DNA to RNA, purified plasmid DNA is linearized by
digestion with XbaI restriction enzyme. After extraction with
phenol-chloroform and precipitation with ethanol, the samples were
suspended in water. Typically, about 1 .mu.g of linear plasmid DNA was
transcribed in reactions (25 .mu.l) with T7 RNA polymerase as specified by
the enzyme manufacturer (Bethesda Research Laboratories), except that the
ribonucleotides and dithiothreitol were increased to lmM and 25 mM,
respectively. RNase inhibitor (RNasin; Promega Biotec) was also included
(1.5 U/ .mu.l). After incubation at 37.degree. C for 1 h, the samples were
extracted with phenolchloroform, precipitated with ethanol, dried under 7
vacuum, and suspended in water (10 .mu.l; estimated concentration, 1
.mu.g/.mu.l).
In vitro translation reactions in reticulocyte extracts were carried out in
a manner analogous to the procedures of D. Shih et al., 30 J. Virol.
472-480 (1979). Typically, 3 to 5 .mu.l of plasmid transcription product
(see above) was used to direct cell-free protein synthesis reactions (30
.mu.l) radiolabeled with [.sup.35 S]methionine (specific activity, 1,100
Ci/mmol; final concentration, 1 Ci/.mu.l). After 40 min. at 30.degree. C,
reactions were stopped by addition of pancreatic RNase and cycloheximide
(to 0.3 mg/ml each).
This technique can be used to produce a single EMC virion protein (as
opposed to the naturally occurring string of EMC proteins). See G. Parks
et al., 60 J. Virol. 376-387 (1986) (not prior art). This opens up the
possibility of research directed to particular EMC proteins. For example,
the availability of large quantities of EMC proteins such as the protease
3C will permit such proteins to be used in assays to screen for drugs that
block the activity of the proteins (and thus block the activity of the
virus). In this regard, a protease splits other compounds. By exposing a
given quantity of such a protein to a possible drug in the presence of a
substance that it usually effects (e.g. cleaves), chromatographic and/or
other techniques can determine which if any compounds inhibit protein
activity. Because many viruses have protease sequences, and because all
picornaviruses appear to have similar protease sequences, the EMC protease
assay may act as a screen for drugs for other viral proteases as well.
This is important because some viruses are very dangerous to work with in
laboratories.
As an alternative, a foreign DNA from poliovirus or another virus, or
another non viral source can be inserted. See e.g. H. Krausslich et al.,
61 J. Virol. 2711-2718 (1987) (not prior art).
It will also be appreciated that while one particular EMC derived enhancer
is shown in the drawings, enhancers can be produced synthetically using
the coding between nucleotides 488 and 834, or using slight variants of
the natural sequence, or using other cardioviruses with similar 5'
non-coding regions. Thus, sequences substantially having the
characteristics of the EMC sequence are intended to be included in the
language "having the characteristics." Further, while polio and EMC
viruses have been referred to as the expressed genes, eukaryotic and viral
translation may be broadly enhanced by this region.
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