CA2022356A1 - Method and materials for producing proteins - Google Patents
Method and materials for producing proteinsInfo
- Publication number
- CA2022356A1 CA2022356A1 CA 2022356 CA2022356A CA2022356A1 CA 2022356 A1 CA2022356 A1 CA 2022356A1 CA 2022356 CA2022356 CA 2022356 CA 2022356 A CA2022356 A CA 2022356A CA 2022356 A1 CA2022356 A1 CA 2022356A1
- Authority
- CA
- Canada
- Prior art keywords
- mrna
- sequence
- estrogen
- coding
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
The invention provides a hybrid mRNA molecule comprising a first RNA sequence coding for a protein or polypeptide of interest operatively linked to a second RNA sequence which is capable of stabilizing the hybrid mRNA in the presence of estrogen and estrogen receptor.
The hybrid mRNA molecule may further comprise a third sequence located between the first and second sequences which codes for a cleavage site so that the protein or polypeptide of interest may be cleaved from unnecessary or interfering amino acid sequences coded for by the second RNA sequence. The invention further provides a recombinant DNA sequence coding for the hybrid mRNA, a vector comprising the recombinant DNA sequence operatively linked to expression control sequences, and a host cell transformed with the vector. Finally, the invention provides a method of stabilizing an mRNA so as to obtain increased production of a protein or polypeptide. The method comprises growing the transformed host cell in the presence of estrogen.
The invention provides a hybrid mRNA molecule comprising a first RNA sequence coding for a protein or polypeptide of interest operatively linked to a second RNA sequence which is capable of stabilizing the hybrid mRNA in the presence of estrogen and estrogen receptor.
The hybrid mRNA molecule may further comprise a third sequence located between the first and second sequences which codes for a cleavage site so that the protein or polypeptide of interest may be cleaved from unnecessary or interfering amino acid sequences coded for by the second RNA sequence. The invention further provides a recombinant DNA sequence coding for the hybrid mRNA, a vector comprising the recombinant DNA sequence operatively linked to expression control sequences, and a host cell transformed with the vector. Finally, the invention provides a method of stabilizing an mRNA so as to obtain increased production of a protein or polypeptide. The method comprises growing the transformed host cell in the presence of estrogen.
Description
~23~
METHOD AND MATERIALS
FOR PRODUCING PROTEINS
The research which forms a basis of this in-vention was supported in part by grant DCB 86-04109 from the National Science Foundation. The U.S. government may have rights in this invention.
~A~RGROUND OF THE INVE~TION
This invention relates to methods and materials for producing proteins and polypeptides by recombinant DNA techniques. In particular, this invention relates to a method and materials useful in stabilizing a messenger RNA (mRNA) to obtain increased production of the protein or polypeptide coded for by the mRNA.
A major goal of the biotechnology industry is the production of large amounts of desired proteins and polypeptides by transformed host cells. To date, vir-tually all efforts to produce systems for the expression of proteins and polypeptides of interest have centered around the production of vectors with efficient promoters or with the capacity to exist at high copy number within cells. Increasing the stability of mRNA transcripts has been largely neglected.
The intracellular level of an mRNA dictates the amount of protein or polypeptide which can be produced by translation of that mRNA. In turn, the level of the mRNA
is regulated by its rate of synthesis and its rate of degradation (its stability). Regulation of the stability of eukaryotic mRNAs is a common control mechanism, and the processes governing the degradation of the more than 20 eukaryotic mRNAs whose stability is known to be regulated are currently attracting considerable attention. See Cleveland and Yen, New Biol., 1, 121-126 ~1989); Brawerman, Ç~ll, 57, 9-lo (1989); Raghow, Trends Biochem. Sci~ , 358-360 (1987); Ross, Sci. Am., 260, 48-55 (1989); Shapiro et al., ~ioEssays, 6, 221-226 (1987); Nielsen and Shapiro, Molecular Endo., 4, 953-57 ( 1990) -It has been reported that the stability of a few mRNA~ is regulated by their 5' untranslated regions.
Morris et al., Proc. Natl. Acad. Sci. USA, 83, 981-985 (19B6); Trei~man, Ç~ll, 42, 889-902 (1985); Dani et al., Proc. Natl. Acad. Sci. USA, 81, 7046-7050 (1984); Eick et al., EMB0 J., 4, 3717-3725 (1985). Indeed, Santos et al. have suggested a role for this region in the estrogen regulation of c-myc mRNA. Santos et al., J. Biol. Chem., 263, 9565-9568 (1988). However, most studies have identified stem and loop structures, A+U-rich sequences and other elements in the 3' untranslated region, and the length of the poly(A) tract available for interaction with the poly(A)-binding protein, as being major determinants of mRNA stability. Mullner and Kuhn, Cell, 53, 815-825 (1988); Pandey and Marzloff, Mol. Cell.
Biol., 7, 4557-4559 (1987); Ross and Kobs, J. Mol.
Biol., 188, 579-593 (1986); Brewer and Ross, Mol. Cell.
Biol., 8, 1697-1708 (1988); Shaw and Kamen, Cell, 46, 659-667 (1986); Carrazana et al., Mol. Cell. Biol~, 8, 2267-2274 (1988); Muschel et al., Mol. Cell. ~iol., 6, 337-341 (1986); Paek and Axel, Mol. Cell. Biol., 7, 1496-1507 (1987); Shapiro et al., Mol. Cell. Biol., 8, 1957-1969 ~1988).
Translation is required for controlling the stability of ~-tubulin mRNA and histone mRNAs. Yen et al., Nature (London), 334, 580-585 (1988); Graves et al., Cell, 48, 615-626 (1987)). Premature translation r~ -. . ~.. ~ iJ f.,; ~3 .,~
termination results in the degradation of triosephosphate isomerase and yeast URA1 and PGK1 mRNAs. Daar and Maquat, Mol. Cell. Biol., 8, 802-813 (1988); Pelsy and Lacroute, Curr. Gçnet., 8, 277-282 (1984); Hoek~a et al., Mol. Cell. ~iol., 7, 2~14-2924 (1987).
Although hormones have been shown to regulate the ~tability of several mRNAs, the absence of functional assays for steroid hormone control of mRNA degradation ha~ greatly hindered studies. As a consequence, the mechanisms controlling mRNA degradation in these systems remain largely unknown.
One mRNA whose synthesis and stability are regulated by a hormone is the mRNA coding for the Xeno~us laevi8 egg yolk precursor protein vitellogenin.
The regulation of vitellogenin gene transcription and control of the cytoplasmic stability of vitellogenin mRNA, not changes in the efficiency of nuclear vitellogenin RNA processing, are responsible for the massive estrogen induction of the hepatic mRNA coding for vitellogenin. Blume et al., UCLA SYmD. New Ser., 52, 259-274 (1987); Brock and Shapiro, J. Biol. Chem., 9, 5449-5455 (1983); Brock and Shapiro, Cell, 34, 207-214 (1983).
The stability of vitellogenin mRNA is greatly increased in the presence of high levels of estradiol-17~. Brock and Sha~iro, Cell, 34, 207-214 (1983). In Xenopus liver fragment cultures, vitellogenin mRNA is essentially stable (with a half-life of more than 500 hours) when estrogen is present. Id. Upon removal of the estrogen, vitellogenin mRNA begins to rapidly degrade with a half-life of 16 hours at 25C or 30 hours at 20C. Blume and Shapiro, Nucleic Acids Res., 17, 9003-9014 (1989). Vitellogenin mRNA undergoing rapid cytoplasmic degradation can be specifically restabilized by the addition of estrogen to the culture medium [Brock and Shapiro, Cell, 34, 207-214 (1983)], indicating that , 2 ~ ~3 ~
vitellogenin mRNA stabilization is a reversible cytoplasmic ef~ect of estrogen. In hepatocytes, estrogen elicits a 20-fold increase in the stability of vitellogenin mRNA and decreases the stability of albumin mRNA by 3-fold without affecting the 16-hour half-life (at 25C) of total poly(A) mRNA. Id.; Reigel et al., Mol. Cell. Endocrinol, 44, 201-209 (1986); Wolffe et al., Eur. J. Biochem., 146, 489-496 (1985). The broad spectrum of estrogen effects on mRNA stability in Xeno~us liver provides a striking example of the specificity and flexibility o~ this type of control.
SUMMARY OF THE INVENTION
The invention provides a hybrid mRNA molecule comprising a first RNA sequence coding for a protein or polypeptide of interest. This first RNA sequence is operatively linked to a second RNA sequence which is capable of stabilizing the hybrid mRNA in the presence of estrogen and estrogen receptor.
The hybrid mRNA molecule may further comprise a third sequence located between, and operatively ~in~ed to, the first and second sequences. The third sequence codss for a cleavage site so that the protein or polypeptide of interest may be cleaved from unnecessary or interfering amino acid sequences coded for by the second RNA sequence.
The invention further provides a recombinant DNA sequence coding for the hybrid mRNA and a vector comprising the recombinant DNA sequence operatively linked to expression control sequences. The invention also provides a host cell transformed with the vector.
Finally, the invention provides a method of stabilizing an mRNA so as to obtain increased production of a protein or polypeptide. The method comprises transforming the host cell of the invention with the vector Df the invention. If the hos' cell does not c~ r~
"~ J ,~ y~
normally produce estrogen receptor, it must also be transformed with a vector coding for estrogen receptor.
Finally, the transformed host cell is grown in the presence of estrogen. Under these conditions, the hybrid mRNA transcribed from the recombinant DNA sequence of the invention is stabilized o that increased production of the protein or polypeptide of interest is obtained.
Indeed, the level of the hybrid mRNAs of the invention can be increased by up to 20-fold relative to other mRNA~ when the transformed host cells of the inven-tion are cultured in the presence of estrogen. Thus, the use of the present invention will greatly decrease the cost of producing many proteins and polypeptides by genetic engineering techniques since the number of cells and the amount of cell culture medium required to produce a given amount of protein or polypeptide will be substan-tially reduced. Further, the protein or polypeptide will be present at a higher concentration relative to other cell proteins, and the number and complexity of the purification procedures required to obtain the protein or polypeptide of interest in usable form will also be reduced. This simplification of the purification procedures will also lower the cost of producing the proteins and polypeptides.
BRIFF DESCRIPTION OF THE DRAWINGS
Figure lA: Diagram of pMV5, MV5 mRNA and the MV5 probe.
Fiqures lB-D: Flow diagrams illustrating the preparation of pMV5.
Fiaures 2A-B: Shows the results of assays of the stabilization of MV5 mRNA by estradiol and the estrogen receptor.
Fiqure 3: Schematic of the MV5 and TK probes and the sizes of the parts of the probes protected by MV5, vitellogenin Bl and TK mRNAs.
,f.~ ~ f9 Fiure 4: Flow diagram illustrating the prepa-ration of pXER.
DETAILED DESCRIPTION OF THE
PRESENTLY PREFERRED EMBODI~ENTS
The first RNA sequence of the hybrid mRNAs of the invention codes for a protein or polypeptide of interest. The protein or polypeptide may be one that is normally made by the host cell (a "homologous" protein or polypeptide) or may be one that i8 nQ~ normally made by the host cell (a "heterologous" protein or polypeptide).
In this manner, even an mRNA coding for a homologous protein or polypeptide may be stabilized and production of the homologous protein or polypeptide enhanced.
Suitable proteins and polypeptides include:
human and other animal hormones such as human insulin, human growth hormone, bovine growth hormone, swine growth hormone, thyroid stimulating hormone, follicle stimulating hormone, vasopressin and prolactin; cell growth factors; the various interferons; hormone recep-tors; oncogenes; blood factors such as Factor VII, Factor VIII, erythropoietin and tissue plasminogen activator;
lymphokine~; globulins such as immunoglobulins; albumins;
endorphins such as beta-endorphin and enkephalin;
enzymes; viral antigens such as influenza antigenic protein and hepatitis core and surface antigens; and other useful proteins and polypeptides.
The second RNA sequence codes for an RNA
sequence which is capable of stabilizing the hybrid mRNA
in the presence of estrogen and estrogen receptor. This second RNA sequence may be derived from any mRNA which is normally stabilized by estrogen and estrogen receptor.
Such mRNAs include those coding for vitellogenin, conalbumin, ovalbumin, and apo VLDL II. The second RNA
sequance may comprise one or more portions of these mRNAs.
METHOD AND MATERIALS
FOR PRODUCING PROTEINS
The research which forms a basis of this in-vention was supported in part by grant DCB 86-04109 from the National Science Foundation. The U.S. government may have rights in this invention.
~A~RGROUND OF THE INVE~TION
This invention relates to methods and materials for producing proteins and polypeptides by recombinant DNA techniques. In particular, this invention relates to a method and materials useful in stabilizing a messenger RNA (mRNA) to obtain increased production of the protein or polypeptide coded for by the mRNA.
A major goal of the biotechnology industry is the production of large amounts of desired proteins and polypeptides by transformed host cells. To date, vir-tually all efforts to produce systems for the expression of proteins and polypeptides of interest have centered around the production of vectors with efficient promoters or with the capacity to exist at high copy number within cells. Increasing the stability of mRNA transcripts has been largely neglected.
The intracellular level of an mRNA dictates the amount of protein or polypeptide which can be produced by translation of that mRNA. In turn, the level of the mRNA
is regulated by its rate of synthesis and its rate of degradation (its stability). Regulation of the stability of eukaryotic mRNAs is a common control mechanism, and the processes governing the degradation of the more than 20 eukaryotic mRNAs whose stability is known to be regulated are currently attracting considerable attention. See Cleveland and Yen, New Biol., 1, 121-126 ~1989); Brawerman, Ç~ll, 57, 9-lo (1989); Raghow, Trends Biochem. Sci~ , 358-360 (1987); Ross, Sci. Am., 260, 48-55 (1989); Shapiro et al., ~ioEssays, 6, 221-226 (1987); Nielsen and Shapiro, Molecular Endo., 4, 953-57 ( 1990) -It has been reported that the stability of a few mRNA~ is regulated by their 5' untranslated regions.
Morris et al., Proc. Natl. Acad. Sci. USA, 83, 981-985 (19B6); Trei~man, Ç~ll, 42, 889-902 (1985); Dani et al., Proc. Natl. Acad. Sci. USA, 81, 7046-7050 (1984); Eick et al., EMB0 J., 4, 3717-3725 (1985). Indeed, Santos et al. have suggested a role for this region in the estrogen regulation of c-myc mRNA. Santos et al., J. Biol. Chem., 263, 9565-9568 (1988). However, most studies have identified stem and loop structures, A+U-rich sequences and other elements in the 3' untranslated region, and the length of the poly(A) tract available for interaction with the poly(A)-binding protein, as being major determinants of mRNA stability. Mullner and Kuhn, Cell, 53, 815-825 (1988); Pandey and Marzloff, Mol. Cell.
Biol., 7, 4557-4559 (1987); Ross and Kobs, J. Mol.
Biol., 188, 579-593 (1986); Brewer and Ross, Mol. Cell.
Biol., 8, 1697-1708 (1988); Shaw and Kamen, Cell, 46, 659-667 (1986); Carrazana et al., Mol. Cell. Biol~, 8, 2267-2274 (1988); Muschel et al., Mol. Cell. ~iol., 6, 337-341 (1986); Paek and Axel, Mol. Cell. Biol., 7, 1496-1507 (1987); Shapiro et al., Mol. Cell. Biol., 8, 1957-1969 ~1988).
Translation is required for controlling the stability of ~-tubulin mRNA and histone mRNAs. Yen et al., Nature (London), 334, 580-585 (1988); Graves et al., Cell, 48, 615-626 (1987)). Premature translation r~ -. . ~.. ~ iJ f.,; ~3 .,~
termination results in the degradation of triosephosphate isomerase and yeast URA1 and PGK1 mRNAs. Daar and Maquat, Mol. Cell. Biol., 8, 802-813 (1988); Pelsy and Lacroute, Curr. Gçnet., 8, 277-282 (1984); Hoek~a et al., Mol. Cell. ~iol., 7, 2~14-2924 (1987).
Although hormones have been shown to regulate the ~tability of several mRNAs, the absence of functional assays for steroid hormone control of mRNA degradation ha~ greatly hindered studies. As a consequence, the mechanisms controlling mRNA degradation in these systems remain largely unknown.
One mRNA whose synthesis and stability are regulated by a hormone is the mRNA coding for the Xeno~us laevi8 egg yolk precursor protein vitellogenin.
The regulation of vitellogenin gene transcription and control of the cytoplasmic stability of vitellogenin mRNA, not changes in the efficiency of nuclear vitellogenin RNA processing, are responsible for the massive estrogen induction of the hepatic mRNA coding for vitellogenin. Blume et al., UCLA SYmD. New Ser., 52, 259-274 (1987); Brock and Shapiro, J. Biol. Chem., 9, 5449-5455 (1983); Brock and Shapiro, Cell, 34, 207-214 (1983).
The stability of vitellogenin mRNA is greatly increased in the presence of high levels of estradiol-17~. Brock and Sha~iro, Cell, 34, 207-214 (1983). In Xenopus liver fragment cultures, vitellogenin mRNA is essentially stable (with a half-life of more than 500 hours) when estrogen is present. Id. Upon removal of the estrogen, vitellogenin mRNA begins to rapidly degrade with a half-life of 16 hours at 25C or 30 hours at 20C. Blume and Shapiro, Nucleic Acids Res., 17, 9003-9014 (1989). Vitellogenin mRNA undergoing rapid cytoplasmic degradation can be specifically restabilized by the addition of estrogen to the culture medium [Brock and Shapiro, Cell, 34, 207-214 (1983)], indicating that , 2 ~ ~3 ~
vitellogenin mRNA stabilization is a reversible cytoplasmic ef~ect of estrogen. In hepatocytes, estrogen elicits a 20-fold increase in the stability of vitellogenin mRNA and decreases the stability of albumin mRNA by 3-fold without affecting the 16-hour half-life (at 25C) of total poly(A) mRNA. Id.; Reigel et al., Mol. Cell. Endocrinol, 44, 201-209 (1986); Wolffe et al., Eur. J. Biochem., 146, 489-496 (1985). The broad spectrum of estrogen effects on mRNA stability in Xeno~us liver provides a striking example of the specificity and flexibility o~ this type of control.
SUMMARY OF THE INVENTION
The invention provides a hybrid mRNA molecule comprising a first RNA sequence coding for a protein or polypeptide of interest. This first RNA sequence is operatively linked to a second RNA sequence which is capable of stabilizing the hybrid mRNA in the presence of estrogen and estrogen receptor.
The hybrid mRNA molecule may further comprise a third sequence located between, and operatively ~in~ed to, the first and second sequences. The third sequence codss for a cleavage site so that the protein or polypeptide of interest may be cleaved from unnecessary or interfering amino acid sequences coded for by the second RNA sequence.
The invention further provides a recombinant DNA sequence coding for the hybrid mRNA and a vector comprising the recombinant DNA sequence operatively linked to expression control sequences. The invention also provides a host cell transformed with the vector.
Finally, the invention provides a method of stabilizing an mRNA so as to obtain increased production of a protein or polypeptide. The method comprises transforming the host cell of the invention with the vector Df the invention. If the hos' cell does not c~ r~
"~ J ,~ y~
normally produce estrogen receptor, it must also be transformed with a vector coding for estrogen receptor.
Finally, the transformed host cell is grown in the presence of estrogen. Under these conditions, the hybrid mRNA transcribed from the recombinant DNA sequence of the invention is stabilized o that increased production of the protein or polypeptide of interest is obtained.
Indeed, the level of the hybrid mRNAs of the invention can be increased by up to 20-fold relative to other mRNA~ when the transformed host cells of the inven-tion are cultured in the presence of estrogen. Thus, the use of the present invention will greatly decrease the cost of producing many proteins and polypeptides by genetic engineering techniques since the number of cells and the amount of cell culture medium required to produce a given amount of protein or polypeptide will be substan-tially reduced. Further, the protein or polypeptide will be present at a higher concentration relative to other cell proteins, and the number and complexity of the purification procedures required to obtain the protein or polypeptide of interest in usable form will also be reduced. This simplification of the purification procedures will also lower the cost of producing the proteins and polypeptides.
BRIFF DESCRIPTION OF THE DRAWINGS
Figure lA: Diagram of pMV5, MV5 mRNA and the MV5 probe.
Fiqures lB-D: Flow diagrams illustrating the preparation of pMV5.
Fiaures 2A-B: Shows the results of assays of the stabilization of MV5 mRNA by estradiol and the estrogen receptor.
Fiqure 3: Schematic of the MV5 and TK probes and the sizes of the parts of the probes protected by MV5, vitellogenin Bl and TK mRNAs.
,f.~ ~ f9 Fiure 4: Flow diagram illustrating the prepa-ration of pXER.
DETAILED DESCRIPTION OF THE
PRESENTLY PREFERRED EMBODI~ENTS
The first RNA sequence of the hybrid mRNAs of the invention codes for a protein or polypeptide of interest. The protein or polypeptide may be one that is normally made by the host cell (a "homologous" protein or polypeptide) or may be one that i8 nQ~ normally made by the host cell (a "heterologous" protein or polypeptide).
In this manner, even an mRNA coding for a homologous protein or polypeptide may be stabilized and production of the homologous protein or polypeptide enhanced.
Suitable proteins and polypeptides include:
human and other animal hormones such as human insulin, human growth hormone, bovine growth hormone, swine growth hormone, thyroid stimulating hormone, follicle stimulating hormone, vasopressin and prolactin; cell growth factors; the various interferons; hormone recep-tors; oncogenes; blood factors such as Factor VII, Factor VIII, erythropoietin and tissue plasminogen activator;
lymphokine~; globulins such as immunoglobulins; albumins;
endorphins such as beta-endorphin and enkephalin;
enzymes; viral antigens such as influenza antigenic protein and hepatitis core and surface antigens; and other useful proteins and polypeptides.
The second RNA sequence codes for an RNA
sequence which is capable of stabilizing the hybrid mRNA
in the presence of estrogen and estrogen receptor. This second RNA sequence may be derived from any mRNA which is normally stabilized by estrogen and estrogen receptor.
Such mRNAs include those coding for vitellogenin, conalbumin, ovalbumin, and apo VLDL II. The second RNA
sequance may comprise one or more portions of these mRNAs.
2~2 ~ ~f 'if..i It preferably comprises one or more portions of a vitellogenin mRNA, most preferably those portions of the Xeno~us laevis vitellogenin Bl mRNA described in Example 1. In Example 1, the X. laevis vitellogenin B1 mRNA sequences used for stabilizing mRNA include the following: a fir~t portion comprising the entire 5' untranslated region (13 nucleotides) linked to 81 nucleotides coding for the 27 amino-terminal amino acids of vitellogenin B1; and a second portion compriaing 270 nucleotides coding for the carboxy-terminal 90 amino acid~ of vitellogenin Bl linked to the entire 3' untranslated region (165 nucleotides). In a hybrid mRNA
aocording to the invention, the first portion of the vitellogenin B1 sequences would be located 5' of the sequence coding for the desired protein or polypeptide, and the second portion would be located 3' of this sequence. Of course, all sequences are operatively linked so that the hybrid mRNA is stabilized in the presence of estrogen and estrogen receptor and is translated to produce a protein or polypeptide of interest having the correct amino acid sequence.
If the second RNA sequence contains coding sequences, the hybrid mRNA may further comprise a third sequence located between the first and second RNA
sequences. The third sequence codes for a chemical or enzymatic cleavage site so that the protein or polypep-tide of interest may be cleaved from any unnecessary or interfering amino acid sequences coded for by the second RNA sequence. In the instance where the second RNA
sequence comprises two parts, two additional sequences coding for cleavage sites may be necessary. For instance, in the case of the vitellogenin Bl mRNA
sequences described above, a third sequence coding for a cleavage site may be included between the first portion of the vitellogenin B1 mRNA sequence and the first RNA
sequence coding for the protein or polypeptide of interest. Also, a fourth sequence coding for a cleavage site may be included between the first RNA se~uence coding for the protein or polypeptide of interest and the ~econd portion of the vitellogenin Bl mRNA sequence.
The cleavage site may be a site for proteolytic cleavage. Many such sites and RNA and DNA sequences coding for th~m are well-known. Alternatively, where the selectQd protein or polypeptide of interest does not contain any methionine residue~, the cleavage site may be methionine (encoded for by an ATG codon in DNA). The protein or polypeptide of interest may then be cleaved from the amino acids coded for by the second RNA sequence at the methionine re8idue by treatment with cyanogen bromide. Gros6, Methods in Enzymoloav, 11, 238-55 (1967).
The hybrid mRNAs of the invention are produced by the transcription of a recombinant DNA sequence that codes for the hybrid mRNA, and the invention also com-prises this recombinant DNA sequence and a vector in which the recombinant DNA sequence is operatively linked to expression control sequences. The recombinant DNA
sequence can be prepared and incorporated into the vector using conventional techniques known to those skilled in the art.
First, the DNA sequences coding for the mRNA
stabilization sequences and for the protein or polypep-tide of interest are isolated. This may be accomplished by constructing a cDNA or a genomic DNA library and screening for the DNA sequence of interest using appro-priate hybridization probes. Of course, many genes and DNA sequences useful in the practice of the invention have already been isolated and cloned and are readily available. For instance, vitellogenin has been cloned (see below), as have many proteins and polypeptides of interest such as the interferons and human insulin.
Further, many desired DNA sequences may be prepared by s~ ~ ~ r~
~ ~J i" ~J ~
chemical synthesis if the DNA or amino acid sequence is known.
The recombinant DNA sequence of the invention is prepared by linking the DNA sequence coding for the mRNA ~tabilization sequences to the DNA sequence coding for the protein or polypeptide of interest. The DNA
sequence coding for the cleavage site, if used, is built into the protein or polypeptide produced by the hybrid mRNA by constructing the recombinant DNA sequence so that it has one or more codons that code for the desired cleavage site located between the DNA sequence(s) encoding the mRNA stabilization sequences and the DNA
sequence encoding the protein or polypeptide of interest.
All of the DNA sequences are operatively linked so that the hybrid mRNA produced by transcribing the recombinant DNA sequence is stabilized by estrogen and estrogen receptor and is translated so as to produce a protein or polypeptide of the proper amino acid sequence.
The invention also includes a vector capable of expressing the protein or polypeptide in an appropriate host. The vector comprises the recombinant DNA molecule linked to appropriate expression control sequences.
Methods of effecting this operative linking, either before or after the recombinant DNA sequence is inserted into the vector, are well known.
Expression control sequences include promoters, activators, enhancers, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation si~nals, and other signals involved with the control of transcription or translation. Expression control sequences suitable for use in the invention are well known. They include sequences known to control the expression of genes of eukaryotic cells, their viruses, or combinations thereof.
~ ~ 2 J D ,~ `~
The vector must contain a promoter and a transcription termination signal, both operatively linked to the recombinant DNA sequence. The promoter may be any DNA sequence that show~ transcriptional activit~ in the host cell and may be derived from genes encoding homologous or heterologous proteins (preferably homo-logous) and either extracellular or intracellular pro-teins, such as thymidine kinases, vitellogenin~, amyla~e~, glycoamyla~e~, protease~, lipases, cellulases and glycolytic enzyme~.
The promoter may be preceded by upstream acti-vator and enhancer sequences. In particular, estrogen response elements which operate as enhancers may be used.
In this manner, the estrogen and estrogen receptor that are used to stabilize the hybrid mR~A would also enhance transcription of the mRNA.
The vector should also have a translation start signal immediately preceding the recombinant DNA
sequence, if the recombinant DNA sequence does not itself begin with such a start signal. There should be no stop si~nal between the start signal and the end of the recombinant DNA sequence.
The vector must also contain one or more origins of replication which allow it to replicate in the host cells. The vector should further include one or more restriction enzvme sites for inserting the recom-binant DNA and other DNA sequences into the vector, and a DNA ~equence coding for a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell ("a selection marker").
Suitable vectors for use in the invention are well known. They include vectors suitable for use in eukaryotic cells such as viral vectors (retroviral vec-tors, vaccinia vectors), other vectors described in the Examples below, and derivatives of these vectors.
In a preferred embodiment, a DNA sequence encoding a signal or si~nal-leader sequence, or a func-tional fragment thereof, i8 included in the recombinant DNA vector between the translation start signal and the portion of the recombinant DNA sequence coding for the protein or polypeptide of interest. A signal or signal-leader sequence is a sequence of amino acids at the amino terminus of a polypeptide or protein which provides for secretlon of the protein or polypeptide from the cell in which it ie produced. Many such signal and signal-leader sequences are known.
By including a DNA sequence encoding a 6ignal or signal-leader amino acid sequence in the vectors of the invention, the protein or polypeptide encoded by the recombinant DNA sequence may be secreted from the cell in which is it produced. Preferably, the signal or signal-leader amino acid sequence is cleaved from the fusion protein during its secretion from the cell. If not, the protein or polypeptide should preferably be cleaved from the signal or signal-leader amino acid sequence after isolation of the fusion protein.
Signal or signal-leader sequences suitable for use in the invention include: signal sequences which are normally part of precursors of proteins or polypeptides such as the precursors of vitellogenin (see below), parathyroid hormone, and interferon (see U.S. patent No.
4,775,622); synthetic signal-leader sequences;
SaccharomYçes cerevisiae alpha factor (see U.S. patents No~. 4,546,082 and 4,870,008); fragments of S. cerevisiae alpha factor; and the yeast BARl secretion system (see U.S. patent No. 4,613,572).
The resulting vector having the recombinant DNA
sequence of the invention thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.
f., ,~ j'l ;'J ~ .,3 j,~
The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity to it of the protein or p~lypeptide of int~rest encoded for by the recombinant DNA sequence, rate of transformation, ease of recovery of the protein or polypeptide of intereat, expression characteristics, biosafety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular recombinant DNA sequence.
Useful hosts for practicing the present invention are eukaryotic cells. It is not believed that prokaryotes can serve as hosts in the present invention.
Preferred host cells are amphibian and mammalian (including human) liver cells, particularly X. laevis liver cells. Most preferred are the XL110 cell line described below and other similar cell lines which can be prepared from X. laevis liver by the method described below for preparation of the XLllO cell line.
If the host cell does not normally produce estrogen receptor, it must be transformed with a vector which codes for estrogen receptor. Genes coding for estrogen receptors have been cloned. See Weiler et al., Molec. Endo., 1, 355-362 (1987~. vectors suitable for transforming host cells so that they produce estrogen receptor may be prepared by methods well known in the art (see discussion above).
Next, the transformed host cell is cultured in the presence of estrogen so that the hybrid mRNA is transcribed and stabilized. In this manner, production of the the desired protein or polypeptide is enhanced.
In culturing the host cells, normal culture medium and conditions may be used. However, it is desirable to use serum in the culture medium which has been treated to remove endogenous estrogen since long-term exposure to ~ ~ 3 ~ t,~
the estrogen-estrogen receptor complex is toxic to cultured cells.
Finally, after fermentation, the protein or polypeptide is purified. Methods of purifying proteins and polypeptides from cultures are well-known in the art.
s In the Examples which follow, standard tech-nique~ ~or working with nucleic acids, such as restric-tion mapping, agarose and acrylamide gel electrophoresis, isolation of DNA fragments from gels, filling in and blunt-ending protruding ends of restriction fragments, li~ation of DNA fragments, preparing plasmids for trans-formation by calf intestinal alkaline phosphatase diges-tion, growth and transformation of E. ÇQli, preparing plasmid minipreps and large-scale plasmid preparations (by the alkaline lysis method) were carried out as des-cribed in Maniatis et al., Molecular Clonina (Cold Spring Harbor Laboratory, New York 1982). DNA sequencing of double-stranded plasmid DNA was done as described in Chen and Seeberg, DNA, 4 165-170 (1985).
Unless otherwise noted, the enzymes used in the following Examples were obtained from Bethesda Research Laboratories. They were used according to the manufacturer's instructions.
EXA~PLE 1: Construction of lasmid pMV5 A hybrid mini-vitellogenin gene (MV5) derived from the X. laevis vitellogenin Bl gene was constructed.
Normally, vitellogenin genes encode mRNAs of over 5,600 nucleotides [Gerber-Huber et al., Nucleiç Acids ~es., 15, 4737-4760 (1987)], but the MV5 gene codes for an mRNA
lacking 5,075 nucleotides of the internal vitellogenin-coding region. The MV5 mRNA transcript contains the following portions of the vitellogenin B1 mRNA: 1) the entire 5' untranslated region (13 nucleotides); 2) 81 ~ 3 .
nucleotides coding for the 27 amino-terminal amino acids, including the signal peptide; 3) 270 nucleotides coding for the carboxy-terminal 90 amino acids; and 4) the entire 3~ untranslated region (165 nucleotides) (see Figure lA). The processing and polyadenylation signals from the vitellogenin Bl gene are positioned in MV5 at the 3' end 80 that the MV5 transcripts will be properly polyadenylated in X~enopus cell~.
The MV5 gene was constructed so that it coded for regions of the vitellogenin mRNA that have been reported to be important in controlling the stability of other mRNAs (see Background). In particular, the 5' and 3' untranslated regions, the signal sequence, short 3' and 5' coding regions, and genomic sequences specifying processing and polyadenylation of vitellogenin mRNA were included. Of course, it could not be predicted in advance whether the resultant mRNA would be stabilized ince the mechanism of mRNA stabilization by hormones is largely unknown.
Plasmid pMV5 was prepared for expression of the MV5 gene (see Figures lB-D). In this plasmid, expression of the MV5 gene is driven by the herpes simplex virus (HSV) thymidine kinase (TK) promoter, a promoter that is efficiently expressed in Xeno~us cells and is not regulated by estrogen. McKnight et al., Cell, 25, 385-398 (1981); see below. The 5' untranslated and coding region6 of MV5 were constructed by cloning four overlapping synthetic oligonucleotides after the TK
promoter 80 as to direct the transcription of an mRNA
with an authentic vitellogenin Bl 5' end. The 3' coding and untranslated regions are from a vitellogenin Bl cDNA
clone and a genomic vitellogenin Bl clone. To maintain translation of the MV5 transcript, the 3' coding region was fused in frame with the 5' coding region. This construct was inserted into the pTZ19R vector. Finally, the simian virus 40 poly(A) region was placed upstream of ~ 15 ~
the MV5 gene to prevent readthrough transcription intothe MV5 gene. The details of the construction of the pMV5 mini-vitellogenin plasmid are as follows.
Fir~t, the 1235 base pair (bp) Hind III to Hind III fragment containing the vitellogenin B1 3' end was excised from the genomic clone Xlv14.3 (see Figure lB).
Clone XIv14.3 was kindly provided by M. Hayward and D.
Lew of the Univer8ity of Illinoi~, Champaign-Urbana, Illinoi~, and wa~ prepared as described in D. Lew, 1988 Ph.D. Thesis, Univer~ity of Illinois, Champaign-Urbana, Illinois. This fragment, was made blunt ended by filling in the sticky ends with Klenow fragment, and was then in~erted into the Hpa I site of pRSV-CAT to yield pRCV11 (see Figure lB). Plasmid pRSV-CAT is available from Bethesda Research Laboratories and Pharmacia.
Next, a 2728 bp Nco I to Pst I fragment from pRCVll was ligated to a 758 bp Bam HI to Nco I fragment from pTKlCO and a 2800 bp Pst I to Bam HI fragment from pTZ19R to form pTCV1 (see Figure lB). Plasmid pTKlC0, was a gift of Keith Yamamoto, University of California, San Francisco, Ca. Its preparation is described in DeFranco and Yamamoto, Molec. Cell. Biol., 6, 993-1001 (1986) (referred to as OTCO in the article). Plasmid pTZ19R was prepared as described in Mead et al., Protein Enq~, 1, 67-74 (1986).
Plasmid pTCV1 was cleaved with Pst I in the SV40 poly(A) region, the sticky ends removed with mung bean nuclease (obtained from Stratagene) and religated to yield pTCV2 (see Figure lC). Then, a 203 bp Pst I to Bgl II fragment containing 3' vitellogenin B1 cDNA from pXlvc56 was inserted into the Pst I to Bgl II sites of pTCV2 to yield pMV1 (see Figure lC). Clone pXIvc56 was kindly provided by M. ~ayward and D. Lew, University of Illinois, Champaign-Urbana, Illinois and was prepared as described in D. Lew, 1988 Ph.D. Thesis, University of Illinois, Champaign-Urbana, Illinois.
16 ~ 2 ?~ ~J ~ ~
~ ext, a 137 bp fragment containing the SV40 early termination signal wa~ excised from p~CV10 with BglII and ~a~I, and the fragment was then in~erted into the Bam HI site of pMVl to form pMV2 (see Figure lC).
Plasmid pRCV10 was derived by the insertion of Bgl II
linkers (obtained f~om ~ew England ~iolabs Inc.) into the Hpa I site of pRSV~CA~ (see Figure lB).
Four overlapping oligonucleotides were synthesize~ on an Applied Biosystems Model 380B DNA
synthesizer using ~-cyanoeth~lphosphoramide technology, and were then hybridi~ed to yield the following two ove~l~pping fragments:
AGCCCTCGAAT~CGCCATCACC~TGAGGGGA~T~A~ACTAGCTCAGCTTCTCGCTCTAG
AcGT~ccG~:AGcTTAAGc~rAGTGGTAcTccccTTAGTAlGATcGAGTccAA
CGGc:AAGT~AAAAG~cAcAATATGA~ccTTTT~cAG~GAGAGcc~GcA
GAaCGAGF~IrCGCCCTTCAC'rTTTC~A~T~'rTATAC~TGGAAAA ~G'rCAC~CTCGC;
These were ~nse~ted into ~he Pst I si~e of pMV2 to create pMV3 (~ee Figure lD). Then, the 27 bp fragment of pMV3 from between the Hae III site 6 bp upstream of the tk mRNA start site ~nd the Stu I site located in the synthetic oligonucleot~de was deleted to yield pMY4 (see ~igure 1~). The sequence of pMV4 was verified by dideoxy sequencing of all ~he junctions and the synthetic seq~ence. Finally, 583 bp of pMV4 from between the Stu I
site 3' of the vitellogenin gene in the genomic region and the ~ind III site were deleted to yield pMV5 (see Figure lD).
TUTfiL P. u3 EXAMPLE 2: Requirement for estrogen and estrogen receptor for MV5 mRNA
stabilization in txansfected cells A. Cell line XLllO cells were transfected with pMV5 and other plasmids as d0~crib~d b~low. XLllO cells are a clonal line of partially de-differentiated X. laevis liver cells.
This cell line was developed as follows. The liver of an adult male Xeno~us laevis, which had never been expoeed to estradiol, was perfused with an ice-cold solution of 8arth X (Barth and Barth, J. Embvol. and ~x~erimental Morholoav, 7, 210-222 (1959)) without calcium, and then with a collagenase solution (Wangh et al., ~evelop. Biol., 70, 479-489 (1979)). The perfused liver was excised, minced and then digested again with collagenase (Folger, et al., J. Biol. Chem., ~, 8908-8914 (1983)). The dissociated cells from one liver were plated into one flask and maintained in medium containing fructose instead of glucose and 20% dialyzed fetal bovine serum (fibroblasts lack the enzymes to convert fructose to glucose). One of the two clones which grew out was recloned and designated XL110 (also referred to as AllOI) cells.
Both the selection and standard growth of XLllO
cell~ is on 0.6 X Higuchi's medium (Higuchi, J. Cell Phvsiol., 75, 65-72 (1969)), containing 20 mM HEPES, pH
7.4. During cell isolation the cells were maintained in 0.6 X Higuchi' medium supplemented with 20% dialyzed fetal bovine serum. The cells were isolated by growth at 20C in sealed flasks in air. In normal growth of the cells, they are maintained in 0.6 X Higuchi's medium supplemented with either 10% fetal bovine serum or 10%
dextran-charcoal treated fetal bovine serum (Eckert and Xatzenellenbogen, J. Biol. Chem., 257, 8840-8846 (1982)).
The cells are harvested by standard trypsin digestion ~ ft ~ rs P`' .~
using trypsin in O. 6 X PBS ( lOmM sodium phosphate, 150 mM
NaCl, pH 7.4) according to the supplier's (GIBCO) directions. Standard plating of the cells is approxima-tely 1:10.
Charcoal-dextran treatment of serum to remove endogenous estrogen was performed as follows. A 500 ml suspension was prepar~d containing 50 grams Norit A
charcoal, 5 grams dextran (Sigma, No. D-4751), 4.4 grams NaCl and remainder water. When ready to treat serum, the charcoal was resuspended thoroughly, and a 25 ml aliquot removed. The charcoal in the aliquot was pelleted at 3000 rpm for 5 minute~ at room temperature. The supernatant was poured off, and the volume brought to 25 ml by the addition of 0.15 M NaCl. The resuspended charcoal solution was added to 500 ml of fetal bovine serum, and the serum was inactivated at 55C for one hour, with swirling of the bottle every 15 minutes.
Next, the serum was aliquoted into sterile 50 ml tubes, and the charcoal was pelleted by centrifugation at 3000 rpm for 20 minutes at room temperature. The supernatants were harvested, and the centrifugation step repeated using fresh tubes. Finally, the serum supernatants were filtered through a 0.2 micron filter and were stored at -20C until needed. The entire procedure should be carried out using sterile solutions and plasticware.
B. Transfection and induction with estrogen The XL110 cells were grown at 20C in 0.6x phenol red-free Higuchi medium containing 10% charcoal-dextran-treated fetal bovine serum. The cells were grown in air in sealed flasks or in Petri dishes sealed with parafilm.
One day prior to transfection, one million cells were seeded into a 100 mm culture disk containing 9 2 ~ 2 ,~ 3 71 ml of medium. At 2-4 hours ~efore transfection, the medium was changed.
DNA was transfected into the XL110 cells by a modification of the calcium phosphate coprecipitation method described in Parker and Stark, J. Virol., 31, 360-369 (1979). To prepare calcium phosphate coprecipitates, approximately 30 ug of the DNA of interest lin TE at 1-3 ug of DNA/ul) was added to 0.5 ml of 2X HBS in a 15 ml conical tube. TE i~ a solutio~ containing 10 mM Tris and 1 mM EDTA, pH 7.6, and 2X HBS is a solution containing 280 mM NaCl, 50 mM HEPES, pH 7.09, and 1.5 mM sodium pho~phate. The DNA solution was mixed on a vortex mixer at moderate speed, and an equal volume of 0.2 M CaC12 was added ~ropwise to the tube. After S-10 minutes to allow the crystals to form, the DNA-CaP04 co-crystals were added to the dish of cells with gentle mixing. The cells were then incubated for 24 hours.
A sterile 15~ solution of glycerol in cell culture medium was prepared. The cells were glycerol shocked by adding 2 ml of this glycerol solution to each plate, and incubating the cells for 3 minutes. The medium was aspirated, and the plates rinsed 2 times with 0.6 X PBS to remove all glycerol.
After glycerol shock, the cells were removed from the plates with trypsin, pooled, replated, and maintained for 72 hours in medium containing either 100 nM estradiol-17~ and 0.1% ethanol or ~n medium containing 0.1~ ethanol alone. The medium was changed at 24-hour intervals. The purpose of pooling and replating the cells was to ensure accurate comparisons between trans-fected cells maintained in the presence and absence of estrogen.
C. Plasmids In addition to pMV5, XLllO cells were sometimes transfected (as described above) with other plasmids as h (i ~' ;.t9 ~pecified below. These other plasmids are described below.
1. Plasmid pXER
Plasmid pXER is a XenoPus estrogen receptor [XER] expression plasmid in which synthesis of XER mRNA
is driven from the herpes virus thymidine kinase (TK) promoter. Plasmid pXER (also somQtimes referred to as pTKXER or pTKXERO) was prepared as shown in Figure 4.
The XER gene was cloned as described in Weiler et al., Molec. Endo., 1, 355-62 (1987). Briefly, a XER
cDNA clone was isolated by screening a lambda gtlO cDNA
library prepared from poly(A) mRNA from the livers of male Xeno~us laevis which had received multiple in~ections of estradiol-17~ (which induces the XER). The isolated clone was designated AXER4. The insert from AXER4 was removed by digestion with EcoRl, and the isolated insert was subcloned into the EcoR1 site of the pGEM3 vector (Promega Biotech). The resulting clone was designated pXER4.4.
Next plasmid pTKXER2 was prepared as described in D. Lew, 1988 Ph.D. Thesis, University of Illinois, Champaign-Urbana, Illinois. First, the CAT gene was removed from pTKICO. As noted above, the vector pTKlCO
was a gift from Keith Yamamoto. Vector pTKlCO contains the herpes virus TK promoter and the chloramphenicol acetyl transferase (CAT) structural gene. A similar vector (pTK-luciferase) is available from the American Type Culture Collection with the luciferase structural gene in place of the CAT gene (Nordeen, Biotechniaues, 6, 454-458 (1988)). Since the CAT gene was removed in the construction of pXER, the identity of the segment to be deleted is not relevant.
To remove the CAT structural gene from pTKlCO, the vector was digested with BglII and HpaI. The protrudin~ ends on the restriction sites were removed by ~ 'J ~ a treatment with mung bean nuclease (Stratagene) as described by the manufacturer.
Plasmid pXER4.4 was digested with Eco R1, and the 2,418 nucleotide Eco Rl fragment, which contains the entire protein coding region of the XER gene, was isolated by gel electrophoresis. The protruding ends on the isolated restriction fragment were filled in by treatment with the Klenow fragment of DNA polymerase.
The XER fragment from pXER4.4 was inserted at the Bgl II/Hpa I junction in the blunt ended TKlC0 plasmid by blunt end ligation. The resulting vector, designated pTKXER2, was characterized by restriction mapping and DNA sequencing.
The remaining steps of the preparation of pXER
are described in T.C. Chang, 1990 Ph.D. Thesis, University of Illinois, Champaign-Urbana, Illinois.
First, plasmid pTKXER2 was digested with Cla I, and the 3,419 base-pair Cla I fragment was isolated by gel electrophoresis. This fragment contains the entire TK
promoter, the TK 5'-untranslated region, the protein coding region of the XER gene and the SV40 polyadenylation signal. The vector pTZ18U (Pharmacia) was digested with Acc I. The Cla I fragment from pTKXER2 was inserted into the Acc I site of pTZ18U by ligation to produce pXER which was characterized by restriction digestion and DNA sequencing.
2. Plasmid TK7 Plasmid pTK7 synthesizes thymidine kinase (TX) mRNA, and this mRNA is used as an internal standard in some experiments. When pTK7 was used, a TK cRNA probe was also used simultaneously with the MV5 cRNA probe in the Sl nuclease protection assay (see below and Figure 3) ~J ~ 3 Plasmid pTK7 was prepared by cloning the 2061 bp Bam HI/Hind III fragment of pJC331 containing the HSV
TK gene into the Bam HI/Hind III sites of pTZ19U (Mead et al., Protein Ena., 1, 67-74 (1986)). Plasmid pJC331 was obtained fro~ Bernard Roizman, The University of Chicago, Chicago, Illinois. It was prepared by cloning 2040 bp Pvu II/Pvu II fragment containing the HSV TK gene into the Hinc II site of pUC9 (Vieira and Messing, Gene, 19, 259 ~1982)). The original TK gene description may be found in Post et al., Cell, ~, 555-565 (1981).
3- ~las~id pTKCAT
Plasmid pTKCAT is a TK promoter-chlor-amphenicol acetyltransferase gene fusion plasmid that was added in some experiments as carrier DNA to maintain a constant TK promoter concentration. This plasmid is the same as pTKlC0 described above.
aocording to the invention, the first portion of the vitellogenin B1 sequences would be located 5' of the sequence coding for the desired protein or polypeptide, and the second portion would be located 3' of this sequence. Of course, all sequences are operatively linked so that the hybrid mRNA is stabilized in the presence of estrogen and estrogen receptor and is translated to produce a protein or polypeptide of interest having the correct amino acid sequence.
If the second RNA sequence contains coding sequences, the hybrid mRNA may further comprise a third sequence located between the first and second RNA
sequences. The third sequence codes for a chemical or enzymatic cleavage site so that the protein or polypep-tide of interest may be cleaved from any unnecessary or interfering amino acid sequences coded for by the second RNA sequence. In the instance where the second RNA
sequence comprises two parts, two additional sequences coding for cleavage sites may be necessary. For instance, in the case of the vitellogenin Bl mRNA
sequences described above, a third sequence coding for a cleavage site may be included between the first portion of the vitellogenin B1 mRNA sequence and the first RNA
sequence coding for the protein or polypeptide of interest. Also, a fourth sequence coding for a cleavage site may be included between the first RNA se~uence coding for the protein or polypeptide of interest and the ~econd portion of the vitellogenin Bl mRNA sequence.
The cleavage site may be a site for proteolytic cleavage. Many such sites and RNA and DNA sequences coding for th~m are well-known. Alternatively, where the selectQd protein or polypeptide of interest does not contain any methionine residue~, the cleavage site may be methionine (encoded for by an ATG codon in DNA). The protein or polypeptide of interest may then be cleaved from the amino acids coded for by the second RNA sequence at the methionine re8idue by treatment with cyanogen bromide. Gros6, Methods in Enzymoloav, 11, 238-55 (1967).
The hybrid mRNAs of the invention are produced by the transcription of a recombinant DNA sequence that codes for the hybrid mRNA, and the invention also com-prises this recombinant DNA sequence and a vector in which the recombinant DNA sequence is operatively linked to expression control sequences. The recombinant DNA
sequence can be prepared and incorporated into the vector using conventional techniques known to those skilled in the art.
First, the DNA sequences coding for the mRNA
stabilization sequences and for the protein or polypep-tide of interest are isolated. This may be accomplished by constructing a cDNA or a genomic DNA library and screening for the DNA sequence of interest using appro-priate hybridization probes. Of course, many genes and DNA sequences useful in the practice of the invention have already been isolated and cloned and are readily available. For instance, vitellogenin has been cloned (see below), as have many proteins and polypeptides of interest such as the interferons and human insulin.
Further, many desired DNA sequences may be prepared by s~ ~ ~ r~
~ ~J i" ~J ~
chemical synthesis if the DNA or amino acid sequence is known.
The recombinant DNA sequence of the invention is prepared by linking the DNA sequence coding for the mRNA ~tabilization sequences to the DNA sequence coding for the protein or polypeptide of interest. The DNA
sequence coding for the cleavage site, if used, is built into the protein or polypeptide produced by the hybrid mRNA by constructing the recombinant DNA sequence so that it has one or more codons that code for the desired cleavage site located between the DNA sequence(s) encoding the mRNA stabilization sequences and the DNA
sequence encoding the protein or polypeptide of interest.
All of the DNA sequences are operatively linked so that the hybrid mRNA produced by transcribing the recombinant DNA sequence is stabilized by estrogen and estrogen receptor and is translated so as to produce a protein or polypeptide of the proper amino acid sequence.
The invention also includes a vector capable of expressing the protein or polypeptide in an appropriate host. The vector comprises the recombinant DNA molecule linked to appropriate expression control sequences.
Methods of effecting this operative linking, either before or after the recombinant DNA sequence is inserted into the vector, are well known.
Expression control sequences include promoters, activators, enhancers, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation si~nals, and other signals involved with the control of transcription or translation. Expression control sequences suitable for use in the invention are well known. They include sequences known to control the expression of genes of eukaryotic cells, their viruses, or combinations thereof.
~ ~ 2 J D ,~ `~
The vector must contain a promoter and a transcription termination signal, both operatively linked to the recombinant DNA sequence. The promoter may be any DNA sequence that show~ transcriptional activit~ in the host cell and may be derived from genes encoding homologous or heterologous proteins (preferably homo-logous) and either extracellular or intracellular pro-teins, such as thymidine kinases, vitellogenin~, amyla~e~, glycoamyla~e~, protease~, lipases, cellulases and glycolytic enzyme~.
The promoter may be preceded by upstream acti-vator and enhancer sequences. In particular, estrogen response elements which operate as enhancers may be used.
In this manner, the estrogen and estrogen receptor that are used to stabilize the hybrid mR~A would also enhance transcription of the mRNA.
The vector should also have a translation start signal immediately preceding the recombinant DNA
sequence, if the recombinant DNA sequence does not itself begin with such a start signal. There should be no stop si~nal between the start signal and the end of the recombinant DNA sequence.
The vector must also contain one or more origins of replication which allow it to replicate in the host cells. The vector should further include one or more restriction enzvme sites for inserting the recom-binant DNA and other DNA sequences into the vector, and a DNA ~equence coding for a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell ("a selection marker").
Suitable vectors for use in the invention are well known. They include vectors suitable for use in eukaryotic cells such as viral vectors (retroviral vec-tors, vaccinia vectors), other vectors described in the Examples below, and derivatives of these vectors.
In a preferred embodiment, a DNA sequence encoding a signal or si~nal-leader sequence, or a func-tional fragment thereof, i8 included in the recombinant DNA vector between the translation start signal and the portion of the recombinant DNA sequence coding for the protein or polypeptide of interest. A signal or signal-leader sequence is a sequence of amino acids at the amino terminus of a polypeptide or protein which provides for secretlon of the protein or polypeptide from the cell in which it ie produced. Many such signal and signal-leader sequences are known.
By including a DNA sequence encoding a 6ignal or signal-leader amino acid sequence in the vectors of the invention, the protein or polypeptide encoded by the recombinant DNA sequence may be secreted from the cell in which is it produced. Preferably, the signal or signal-leader amino acid sequence is cleaved from the fusion protein during its secretion from the cell. If not, the protein or polypeptide should preferably be cleaved from the signal or signal-leader amino acid sequence after isolation of the fusion protein.
Signal or signal-leader sequences suitable for use in the invention include: signal sequences which are normally part of precursors of proteins or polypeptides such as the precursors of vitellogenin (see below), parathyroid hormone, and interferon (see U.S. patent No.
4,775,622); synthetic signal-leader sequences;
SaccharomYçes cerevisiae alpha factor (see U.S. patents No~. 4,546,082 and 4,870,008); fragments of S. cerevisiae alpha factor; and the yeast BARl secretion system (see U.S. patent No. 4,613,572).
The resulting vector having the recombinant DNA
sequence of the invention thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.
f., ,~ j'l ;'J ~ .,3 j,~
The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity to it of the protein or p~lypeptide of int~rest encoded for by the recombinant DNA sequence, rate of transformation, ease of recovery of the protein or polypeptide of intereat, expression characteristics, biosafety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular recombinant DNA sequence.
Useful hosts for practicing the present invention are eukaryotic cells. It is not believed that prokaryotes can serve as hosts in the present invention.
Preferred host cells are amphibian and mammalian (including human) liver cells, particularly X. laevis liver cells. Most preferred are the XL110 cell line described below and other similar cell lines which can be prepared from X. laevis liver by the method described below for preparation of the XLllO cell line.
If the host cell does not normally produce estrogen receptor, it must be transformed with a vector which codes for estrogen receptor. Genes coding for estrogen receptors have been cloned. See Weiler et al., Molec. Endo., 1, 355-362 (1987~. vectors suitable for transforming host cells so that they produce estrogen receptor may be prepared by methods well known in the art (see discussion above).
Next, the transformed host cell is cultured in the presence of estrogen so that the hybrid mRNA is transcribed and stabilized. In this manner, production of the the desired protein or polypeptide is enhanced.
In culturing the host cells, normal culture medium and conditions may be used. However, it is desirable to use serum in the culture medium which has been treated to remove endogenous estrogen since long-term exposure to ~ ~ 3 ~ t,~
the estrogen-estrogen receptor complex is toxic to cultured cells.
Finally, after fermentation, the protein or polypeptide is purified. Methods of purifying proteins and polypeptides from cultures are well-known in the art.
s In the Examples which follow, standard tech-nique~ ~or working with nucleic acids, such as restric-tion mapping, agarose and acrylamide gel electrophoresis, isolation of DNA fragments from gels, filling in and blunt-ending protruding ends of restriction fragments, li~ation of DNA fragments, preparing plasmids for trans-formation by calf intestinal alkaline phosphatase diges-tion, growth and transformation of E. ÇQli, preparing plasmid minipreps and large-scale plasmid preparations (by the alkaline lysis method) were carried out as des-cribed in Maniatis et al., Molecular Clonina (Cold Spring Harbor Laboratory, New York 1982). DNA sequencing of double-stranded plasmid DNA was done as described in Chen and Seeberg, DNA, 4 165-170 (1985).
Unless otherwise noted, the enzymes used in the following Examples were obtained from Bethesda Research Laboratories. They were used according to the manufacturer's instructions.
EXA~PLE 1: Construction of lasmid pMV5 A hybrid mini-vitellogenin gene (MV5) derived from the X. laevis vitellogenin Bl gene was constructed.
Normally, vitellogenin genes encode mRNAs of over 5,600 nucleotides [Gerber-Huber et al., Nucleiç Acids ~es., 15, 4737-4760 (1987)], but the MV5 gene codes for an mRNA
lacking 5,075 nucleotides of the internal vitellogenin-coding region. The MV5 mRNA transcript contains the following portions of the vitellogenin B1 mRNA: 1) the entire 5' untranslated region (13 nucleotides); 2) 81 ~ 3 .
nucleotides coding for the 27 amino-terminal amino acids, including the signal peptide; 3) 270 nucleotides coding for the carboxy-terminal 90 amino acids; and 4) the entire 3~ untranslated region (165 nucleotides) (see Figure lA). The processing and polyadenylation signals from the vitellogenin Bl gene are positioned in MV5 at the 3' end 80 that the MV5 transcripts will be properly polyadenylated in X~enopus cell~.
The MV5 gene was constructed so that it coded for regions of the vitellogenin mRNA that have been reported to be important in controlling the stability of other mRNAs (see Background). In particular, the 5' and 3' untranslated regions, the signal sequence, short 3' and 5' coding regions, and genomic sequences specifying processing and polyadenylation of vitellogenin mRNA were included. Of course, it could not be predicted in advance whether the resultant mRNA would be stabilized ince the mechanism of mRNA stabilization by hormones is largely unknown.
Plasmid pMV5 was prepared for expression of the MV5 gene (see Figures lB-D). In this plasmid, expression of the MV5 gene is driven by the herpes simplex virus (HSV) thymidine kinase (TK) promoter, a promoter that is efficiently expressed in Xeno~us cells and is not regulated by estrogen. McKnight et al., Cell, 25, 385-398 (1981); see below. The 5' untranslated and coding region6 of MV5 were constructed by cloning four overlapping synthetic oligonucleotides after the TK
promoter 80 as to direct the transcription of an mRNA
with an authentic vitellogenin Bl 5' end. The 3' coding and untranslated regions are from a vitellogenin Bl cDNA
clone and a genomic vitellogenin Bl clone. To maintain translation of the MV5 transcript, the 3' coding region was fused in frame with the 5' coding region. This construct was inserted into the pTZ19R vector. Finally, the simian virus 40 poly(A) region was placed upstream of ~ 15 ~
the MV5 gene to prevent readthrough transcription intothe MV5 gene. The details of the construction of the pMV5 mini-vitellogenin plasmid are as follows.
Fir~t, the 1235 base pair (bp) Hind III to Hind III fragment containing the vitellogenin B1 3' end was excised from the genomic clone Xlv14.3 (see Figure lB).
Clone XIv14.3 was kindly provided by M. Hayward and D.
Lew of the Univer8ity of Illinoi~, Champaign-Urbana, Illinoi~, and wa~ prepared as described in D. Lew, 1988 Ph.D. Thesis, Univer~ity of Illinois, Champaign-Urbana, Illinois. This fragment, was made blunt ended by filling in the sticky ends with Klenow fragment, and was then in~erted into the Hpa I site of pRSV-CAT to yield pRCV11 (see Figure lB). Plasmid pRSV-CAT is available from Bethesda Research Laboratories and Pharmacia.
Next, a 2728 bp Nco I to Pst I fragment from pRCVll was ligated to a 758 bp Bam HI to Nco I fragment from pTKlCO and a 2800 bp Pst I to Bam HI fragment from pTZ19R to form pTCV1 (see Figure lB). Plasmid pTKlC0, was a gift of Keith Yamamoto, University of California, San Francisco, Ca. Its preparation is described in DeFranco and Yamamoto, Molec. Cell. Biol., 6, 993-1001 (1986) (referred to as OTCO in the article). Plasmid pTZ19R was prepared as described in Mead et al., Protein Enq~, 1, 67-74 (1986).
Plasmid pTCV1 was cleaved with Pst I in the SV40 poly(A) region, the sticky ends removed with mung bean nuclease (obtained from Stratagene) and religated to yield pTCV2 (see Figure lC). Then, a 203 bp Pst I to Bgl II fragment containing 3' vitellogenin B1 cDNA from pXlvc56 was inserted into the Pst I to Bgl II sites of pTCV2 to yield pMV1 (see Figure lC). Clone pXIvc56 was kindly provided by M. ~ayward and D. Lew, University of Illinois, Champaign-Urbana, Illinois and was prepared as described in D. Lew, 1988 Ph.D. Thesis, University of Illinois, Champaign-Urbana, Illinois.
16 ~ 2 ?~ ~J ~ ~
~ ext, a 137 bp fragment containing the SV40 early termination signal wa~ excised from p~CV10 with BglII and ~a~I, and the fragment was then in~erted into the Bam HI site of pMVl to form pMV2 (see Figure lC).
Plasmid pRCV10 was derived by the insertion of Bgl II
linkers (obtained f~om ~ew England ~iolabs Inc.) into the Hpa I site of pRSV~CA~ (see Figure lB).
Four overlapping oligonucleotides were synthesize~ on an Applied Biosystems Model 380B DNA
synthesizer using ~-cyanoeth~lphosphoramide technology, and were then hybridi~ed to yield the following two ove~l~pping fragments:
AGCCCTCGAAT~CGCCATCACC~TGAGGGGA~T~A~ACTAGCTCAGCTTCTCGCTCTAG
AcGT~ccG~:AGcTTAAGc~rAGTGGTAcTccccTTAGTAlGATcGAGTccAA
CGGc:AAGT~AAAAG~cAcAATATGA~ccTTTT~cAG~GAGAGcc~GcA
GAaCGAGF~IrCGCCCTTCAC'rTTTC~A~T~'rTATAC~TGGAAAA ~G'rCAC~CTCGC;
These were ~nse~ted into ~he Pst I si~e of pMV2 to create pMV3 (~ee Figure lD). Then, the 27 bp fragment of pMV3 from between the Hae III site 6 bp upstream of the tk mRNA start site ~nd the Stu I site located in the synthetic oligonucleot~de was deleted to yield pMY4 (see ~igure 1~). The sequence of pMV4 was verified by dideoxy sequencing of all ~he junctions and the synthetic seq~ence. Finally, 583 bp of pMV4 from between the Stu I
site 3' of the vitellogenin gene in the genomic region and the ~ind III site were deleted to yield pMV5 (see Figure lD).
TUTfiL P. u3 EXAMPLE 2: Requirement for estrogen and estrogen receptor for MV5 mRNA
stabilization in txansfected cells A. Cell line XLllO cells were transfected with pMV5 and other plasmids as d0~crib~d b~low. XLllO cells are a clonal line of partially de-differentiated X. laevis liver cells.
This cell line was developed as follows. The liver of an adult male Xeno~us laevis, which had never been expoeed to estradiol, was perfused with an ice-cold solution of 8arth X (Barth and Barth, J. Embvol. and ~x~erimental Morholoav, 7, 210-222 (1959)) without calcium, and then with a collagenase solution (Wangh et al., ~evelop. Biol., 70, 479-489 (1979)). The perfused liver was excised, minced and then digested again with collagenase (Folger, et al., J. Biol. Chem., ~, 8908-8914 (1983)). The dissociated cells from one liver were plated into one flask and maintained in medium containing fructose instead of glucose and 20% dialyzed fetal bovine serum (fibroblasts lack the enzymes to convert fructose to glucose). One of the two clones which grew out was recloned and designated XL110 (also referred to as AllOI) cells.
Both the selection and standard growth of XLllO
cell~ is on 0.6 X Higuchi's medium (Higuchi, J. Cell Phvsiol., 75, 65-72 (1969)), containing 20 mM HEPES, pH
7.4. During cell isolation the cells were maintained in 0.6 X Higuchi' medium supplemented with 20% dialyzed fetal bovine serum. The cells were isolated by growth at 20C in sealed flasks in air. In normal growth of the cells, they are maintained in 0.6 X Higuchi's medium supplemented with either 10% fetal bovine serum or 10%
dextran-charcoal treated fetal bovine serum (Eckert and Xatzenellenbogen, J. Biol. Chem., 257, 8840-8846 (1982)).
The cells are harvested by standard trypsin digestion ~ ft ~ rs P`' .~
using trypsin in O. 6 X PBS ( lOmM sodium phosphate, 150 mM
NaCl, pH 7.4) according to the supplier's (GIBCO) directions. Standard plating of the cells is approxima-tely 1:10.
Charcoal-dextran treatment of serum to remove endogenous estrogen was performed as follows. A 500 ml suspension was prepar~d containing 50 grams Norit A
charcoal, 5 grams dextran (Sigma, No. D-4751), 4.4 grams NaCl and remainder water. When ready to treat serum, the charcoal was resuspended thoroughly, and a 25 ml aliquot removed. The charcoal in the aliquot was pelleted at 3000 rpm for 5 minute~ at room temperature. The supernatant was poured off, and the volume brought to 25 ml by the addition of 0.15 M NaCl. The resuspended charcoal solution was added to 500 ml of fetal bovine serum, and the serum was inactivated at 55C for one hour, with swirling of the bottle every 15 minutes.
Next, the serum was aliquoted into sterile 50 ml tubes, and the charcoal was pelleted by centrifugation at 3000 rpm for 20 minutes at room temperature. The supernatants were harvested, and the centrifugation step repeated using fresh tubes. Finally, the serum supernatants were filtered through a 0.2 micron filter and were stored at -20C until needed. The entire procedure should be carried out using sterile solutions and plasticware.
B. Transfection and induction with estrogen The XL110 cells were grown at 20C in 0.6x phenol red-free Higuchi medium containing 10% charcoal-dextran-treated fetal bovine serum. The cells were grown in air in sealed flasks or in Petri dishes sealed with parafilm.
One day prior to transfection, one million cells were seeded into a 100 mm culture disk containing 9 2 ~ 2 ,~ 3 71 ml of medium. At 2-4 hours ~efore transfection, the medium was changed.
DNA was transfected into the XL110 cells by a modification of the calcium phosphate coprecipitation method described in Parker and Stark, J. Virol., 31, 360-369 (1979). To prepare calcium phosphate coprecipitates, approximately 30 ug of the DNA of interest lin TE at 1-3 ug of DNA/ul) was added to 0.5 ml of 2X HBS in a 15 ml conical tube. TE i~ a solutio~ containing 10 mM Tris and 1 mM EDTA, pH 7.6, and 2X HBS is a solution containing 280 mM NaCl, 50 mM HEPES, pH 7.09, and 1.5 mM sodium pho~phate. The DNA solution was mixed on a vortex mixer at moderate speed, and an equal volume of 0.2 M CaC12 was added ~ropwise to the tube. After S-10 minutes to allow the crystals to form, the DNA-CaP04 co-crystals were added to the dish of cells with gentle mixing. The cells were then incubated for 24 hours.
A sterile 15~ solution of glycerol in cell culture medium was prepared. The cells were glycerol shocked by adding 2 ml of this glycerol solution to each plate, and incubating the cells for 3 minutes. The medium was aspirated, and the plates rinsed 2 times with 0.6 X PBS to remove all glycerol.
After glycerol shock, the cells were removed from the plates with trypsin, pooled, replated, and maintained for 72 hours in medium containing either 100 nM estradiol-17~ and 0.1% ethanol or ~n medium containing 0.1~ ethanol alone. The medium was changed at 24-hour intervals. The purpose of pooling and replating the cells was to ensure accurate comparisons between trans-fected cells maintained in the presence and absence of estrogen.
C. Plasmids In addition to pMV5, XLllO cells were sometimes transfected (as described above) with other plasmids as h (i ~' ;.t9 ~pecified below. These other plasmids are described below.
1. Plasmid pXER
Plasmid pXER is a XenoPus estrogen receptor [XER] expression plasmid in which synthesis of XER mRNA
is driven from the herpes virus thymidine kinase (TK) promoter. Plasmid pXER (also somQtimes referred to as pTKXER or pTKXERO) was prepared as shown in Figure 4.
The XER gene was cloned as described in Weiler et al., Molec. Endo., 1, 355-62 (1987). Briefly, a XER
cDNA clone was isolated by screening a lambda gtlO cDNA
library prepared from poly(A) mRNA from the livers of male Xeno~us laevis which had received multiple in~ections of estradiol-17~ (which induces the XER). The isolated clone was designated AXER4. The insert from AXER4 was removed by digestion with EcoRl, and the isolated insert was subcloned into the EcoR1 site of the pGEM3 vector (Promega Biotech). The resulting clone was designated pXER4.4.
Next plasmid pTKXER2 was prepared as described in D. Lew, 1988 Ph.D. Thesis, University of Illinois, Champaign-Urbana, Illinois. First, the CAT gene was removed from pTKICO. As noted above, the vector pTKlCO
was a gift from Keith Yamamoto. Vector pTKlCO contains the herpes virus TK promoter and the chloramphenicol acetyl transferase (CAT) structural gene. A similar vector (pTK-luciferase) is available from the American Type Culture Collection with the luciferase structural gene in place of the CAT gene (Nordeen, Biotechniaues, 6, 454-458 (1988)). Since the CAT gene was removed in the construction of pXER, the identity of the segment to be deleted is not relevant.
To remove the CAT structural gene from pTKlCO, the vector was digested with BglII and HpaI. The protrudin~ ends on the restriction sites were removed by ~ 'J ~ a treatment with mung bean nuclease (Stratagene) as described by the manufacturer.
Plasmid pXER4.4 was digested with Eco R1, and the 2,418 nucleotide Eco Rl fragment, which contains the entire protein coding region of the XER gene, was isolated by gel electrophoresis. The protruding ends on the isolated restriction fragment were filled in by treatment with the Klenow fragment of DNA polymerase.
The XER fragment from pXER4.4 was inserted at the Bgl II/Hpa I junction in the blunt ended TKlC0 plasmid by blunt end ligation. The resulting vector, designated pTKXER2, was characterized by restriction mapping and DNA sequencing.
The remaining steps of the preparation of pXER
are described in T.C. Chang, 1990 Ph.D. Thesis, University of Illinois, Champaign-Urbana, Illinois.
First, plasmid pTKXER2 was digested with Cla I, and the 3,419 base-pair Cla I fragment was isolated by gel electrophoresis. This fragment contains the entire TK
promoter, the TK 5'-untranslated region, the protein coding region of the XER gene and the SV40 polyadenylation signal. The vector pTZ18U (Pharmacia) was digested with Acc I. The Cla I fragment from pTKXER2 was inserted into the Acc I site of pTZ18U by ligation to produce pXER which was characterized by restriction digestion and DNA sequencing.
2. Plasmid TK7 Plasmid pTK7 synthesizes thymidine kinase (TX) mRNA, and this mRNA is used as an internal standard in some experiments. When pTK7 was used, a TK cRNA probe was also used simultaneously with the MV5 cRNA probe in the Sl nuclease protection assay (see below and Figure 3) ~J ~ 3 Plasmid pTK7 was prepared by cloning the 2061 bp Bam HI/Hind III fragment of pJC331 containing the HSV
TK gene into the Bam HI/Hind III sites of pTZ19U (Mead et al., Protein Ena., 1, 67-74 (1986)). Plasmid pJC331 was obtained fro~ Bernard Roizman, The University of Chicago, Chicago, Illinois. It was prepared by cloning 2040 bp Pvu II/Pvu II fragment containing the HSV TK gene into the Hinc II site of pUC9 (Vieira and Messing, Gene, 19, 259 ~1982)). The original TK gene description may be found in Post et al., Cell, ~, 555-565 (1981).
3- ~las~id pTKCAT
Plasmid pTKCAT is a TK promoter-chlor-amphenicol acetyltransferase gene fusion plasmid that was added in some experiments as carrier DNA to maintain a constant TK promoter concentration. This plasmid is the same as pTKlC0 described above.
4. TK-luciferase vector This vector was described above. As noted above, it is available from the American Type Culture Collection, and its preparation is described in Nordeen, Biot~chniques, 6, 454-458 (1988).
5. Plasmid pTK-~-qal.
Plasmid pTK-~-gal is a TK promoter-~-galactosidase gene fusion plasmid. It was constructed by inserting the TK promoter in place of the SV40 promoter in pCHllO. This was accomplished by digesting pCHllO
with KpnI and PvuII and blunt ending the sites by mung bean digestion. The TK promoter was excised ~rom pXER by digestion with EcoRI and PstI. The ends were made blunt by mung bean digestion, and the fragments were joined by blunt end ligation. Plasmid pCHllO is a SV40-~-galactosidase fusion plasmid available from Pharmacia.
`
D. E~ isolation RNA was isolated from transfected XL110 cells at the indicated times after glycerol shock by the guanidine thiocyanate-phenol-chloroform method which is described in Chromczynski and Sacchi, Anal. Biochem., 162, 156-159 (1987). ~enopus liver RNA, isolated from uninduced livers and from livers induced for 12 days with estradiol, was isolated as previoualy described in Shapiro and Bak~r, J. Biol. Chem., 252, 5244-5250 (1977).
E. Solution hybridization and S1 nuclease protection assay Solution hybridization and the S1 nuclease protection assay were performed as descrîbed previously in Salton et al., Mol. Endocrinol., 2, 1033-1042 (1988), with the following modifications. A 2-ng sample of 32p_ labeled cRNA probe (2.7 x 106 cpm) was hybridized to the indicated amount of RNA at 50C overnight. S1 nuclease (1,000 U; Pharmacia, Inc.) was used for digestion at 43C
for 90 min.
After S1 nuclease digestion, samples were electrophoresed in a 4% polyacrylamide gel, using the 1-kb DNA ladder (Bethesda Research Laboratories) as a molecular size standard. Autoradiograms were scanned by using an LKB UltroScan XL densitometer.
Figure 3 presents a schematic of the probes used in the S1 nuclease protection assays and the sizes of the parts of the probes protected by MV5, vitellogenin B1, and TK m~NAs. The 1036-nucleotide MV5 RNA probe used to detect MV5 mRNA in the S1 nuclease protection assay is also shown in Figure lA above the pMV5 construction. The probes were synthesized as described in Salton et al., Mol. Endoc~inol., 2, 1033-42 (1988).
~?.~
F. Results The results of the transfections and the Sl nuclease as~ays are shown in Figures 2A-B and in Tables 1 and 2~ In Figure 2A, the results are shown of an assay in which four plates of XL110 cells were each transfected with 20 ~g of pMV5 and 20 ~g of pXER. RNA isolated from these cell~ was analyzed by the S1 nuclease protection assay using only the MV5 cRNA probe (see Figures lA and 3).
In Figure 2A, the lanes contained the following materials: Lanes 1 and 2---30 ~g of RNA isolated from transfected XLllO cells grown in the absence (Lane 1) or presence (Lane 2) of 100 nM estradiol-17~; Lane 3---10 ~g of RNA from uninduced male Xenopus liver; Lane 4---1 ng of RNA from Xenopus liver induced for 12 days with estrogen plus 10 ~g of uninduced Xenopus liver RNA.
In Figure 2B, the RNA analyzed was from cells transfected as follows: Lanes 1 and 2---10 ~g each of pMV5, pXER, and pTK7; Lanes 3 and 4---10 ~g each of pMV5, pTX7, and pTKCAT; Lane 5---50 ng of HSV-infected Vero cell RNA (obtained from Bernard Roizman, The University of Chicago Medical School, Chicago, Illinois) plus 10 ~g of uninduced Xenopus liver RNA; Lane 6---1 ng of RNA from a Xeno~us liver induced for 12 days with estradiol plus 10 ~g of uninduced Xenopus RNA; Lane 7, 10 ~g of RNA from uninduced male Xeno~us liver. The RNA electrophoresed in Lanes 1 and 3 was from cultures grown for 72 hours in the absence of hormone, and that in Lanes 2 and 4 from cultures grown for 72 hours in 100 nM estradiol-17~. The autoradiograms for Lanes 1, 2, 5, 6, and 7 were exposed for 3 days; those for Lanes 3 and 4 were exposed for 22 hours.
Table 1 presents the MV5 mRNA content found in transfected XLllO cells cultured in the presence of 100 nM estradiol-17~ relative to the MV5 mRNA content in cells cultured in the absence of hormone. The MV5 mRNA
~ ~ 2,, ~ 1 ~
content is expressed as mean + standard error of the mean, and the figures given are the means of nine independent transfection experiments. In each experi-ment, X~110 cells were transfected with 10 or 20 ~g of pMV5 and an equal amount of pXER. RNA isolated from the transfected cells was analyzed by the Sl nuclease pro-tection assay u6ing only the MV5 cRNA probe (see Figures lA and 3).
Control of MV5 mRNA content by estradiol Relative content of MV5 mRNA
Culture Condition (mean + SEM) No hormone 100 100 nN estradiol 164 + 13 Shown in Table 2 are the results of co-transfecting XLllO cells with 10 ~g each pMV5, pTK7 and pXER or with 10 ~g each pMV5, pTK7 and pTKCAT. After 72 hour~, RNA was isolated from the transfected cells, and the levels of MV5 and TK mRNAs were simultaneously determined by the S1 nuclease protection assay. The ratio of MV5 mRNA content to TK mRNA content was used to calculate the content of MV5 mRNA relative to that in cells transfected with pMV5, pXER and pTK7 and grown in no hormone. Plasmid pTK7 was used to provide an internal Rtandard that would correct for any potential estrogen induction of transcription from the TK promoter. When ~ 2 ~Vi w ~ ~ ~
pXER was not used, plasmid pTKCAT was used to keep the level of TK promoter the same in all cells.
Control of MV5 mRNA content by estradiol and estrogen receptor Relative content of MV5 mRNA
DNA~ Culture Condition ~mean ~ SEM) pMV5, pXER No hormone 100 pMV5, pXER 100 nM estradiol 163 + 19 p~V5, pTKCAT No hormone 62 + 12 pMV5, pTKCAT 100 nM estradiol 72 + 12 aAll cells were also transfected with pTK7.
G. Discussion of results 1. Production of MV5 mRNA
Cotransfection of pMV5 and pXER into Xenopus XL110 liver cells resulted in the production of MV5 mRNA.
A 534-nucleotide region of the MV5 cRNA probe, corresponding to the 529-nucleotide MV5 mRNA and the first 5 nucleotides of the poly(A) tail, was protected in the S1 nuclease assay (see Fig. 2A and 2B). Uninduced Xeno~us liver RNA (see Fig. 2A, lane 3; Fig. 2B, lane 7) and mock-transfected XL110 cell RNA (data not shown) produced no protected bands in this size range. Vitel-logenin mRNA from estradiol-induced Xenopus liver pro-tected a 440-nucleotide band, corresponding to the 3' 435 nucleotides of vitellogenin mRNA and the first 5 2 ~ . ~o~ ~i c ~ . ~
nucleotides of the poly(A) tail (see Fig. 2A, lane 4;
Fig. 2B, lane 6). The small 90-nucleotide fragment corresponding to the region of the probe protected by the 5' end o~ full-length vitellogenin mRNA was not seen because it was run off the gel.
2. Role of estrogen in stabilization of MV5 mRNA
To test whether the MV5 mRNA could be stabil-ized by estrogen, pMV5 and pXER were transfected into XLllO cells, and the cells were maintained for 72 hours in the presence or absence of estrogen. MV5 m~NA was stabilized by eRtrogen (see Fig. 2A, Lanes 1 and 2).
When the data from nine separate experiments were aver-aged, the MV5 mRNA content of cells maintained in estradiol was increased by 64 + 13% (see Table 1) com-pared to cells grown in the absence of hormone.
The relative level of MV5 mRNA expected after estrogen stabilization can be calculated by using data obtained from studies of the stability of full-length vitellogenin mRNA in Xenopus liver fragment cultures (Brock and Shapiro, Cell, 34, 207-214 (1983); Blume and Shapiro, Nucleic Acids Res., 17, 9003-9014 (1989)) and from transfection data on the accumulation of MV5 mRNA
and chloramphenicol acetyltransferase enzymatic activity at various times after transfection. Those studies indicate that at 20C there is no significant transcription for 12 hours after transfection into XLllo cells and that transcription for the next 12 hours is at a reduced rate (data not shown). Assuming that transcription then occurs at a constant rate between 24 and 72 hours post-transfection an~ that the MV5 mRNA
exhibits a half-life of 30 hours at 20C in the absence of estrogen and 500 hours (infinite in this calculation) in the presence of estrogen, then we would expect a 1.68-fold increase in MV5 mRNA content due to stabilization by ~J V 2 ~ ~.
estrogen 72 hours after transfection. This value is in excellent agreement with the average 1.64-fold increase in MV5 mRNA levels that was observed (see Table 1).
Because of the relatively long half-life of vitellogenin mRNA in the absence of estrogen, even if transcription of the MV5 gene occurred at a constant rate throughout the 72 hours, estrogen stabilization would result in only a 1.9-fold increa~e in NV5 mRNA content.
Although these data provide strong support for the view that the estradiol-estrogen receptor complex regulates the stability of the MV5 mRNA, they do not explicitly exclude the possibility that this complex induce~ increased transcription of the MV5 gene. Direct evidence that transcription of the TK promoter used in the pMV5 vector is not regulated by estrogen was obtained from experiments in which the same TK promoter in a TK-luciferase vector and plasmid pXER were cotransfected into XL110 cells. In 16 transient cotransfection experiments carried out under conditions similar to those described above, the average level of expression of the TK-luciferase plasmid was 665 luminescence units per ~g in the absence of estradiol and 670 luminescence units per ~g in the presence of estradiol. These values differ by <1%. This result provides compelling evidence that expression of the TK promoter is not induced by the estradiol-XER complex.
The related possibility that the vitellogenin DNA sequence cloned into the pMV5 vector contains an estrogen response element (ERE) that functions as a 3' enhancer is excluded by examination of the sequence of pMV5. The entire nucleotide sequence of the MV5 DNA and the 3'-flanking genomic fragment inserted into the pMV5 clone has been determined. There is no sequence either identical to the consensus ERE or diverging from the ERE
by one, two, or even three nucleotides. All known functional EREs contain zero to two nucleotide changes , ~J ~ ~F"~J 3 c ~ ~
relative to the consensus ERE, indicating that there is no functional estrogen receptor-binding site in the MV5 DNA and its 3'-flanking sequence.
Therefore, three lines of evidence support the conclusion that the estradiol-estrogen receptor complex stabilized MV5 mRNA: (1) the failure of the estradiol-Qstrog~n receptor complex to induce transcription from the TK promoter used in the pMV5 plasmid; (2) the absence of any sequence related to the ERE with the potential to serv2 a~ a 3' transcription enhancer and estrogen receptor-binding site in the MV5 DNA and its flanking DNA; and (3) the excellent agreement between the observed level of MV5 mRNA and the predicted level for an mRNA
undergoing estrogen-mediated stabilization.
3. Requirement for estrogen recetor for MV5 mRNA stabilization In the experiments described above, high levels of estrogen receptor were synthesized from the transfected pXER plasmid. To determine whether the estrogen receptor is required for stabilization of MV5 mRNA, cells were transfected with pMV5 and pXER or with pMV5 and pTKCAT. When pMV5 and pXER were cotransfected, MV5 mRNA was stabilized by estrogen (see Fig. 2B, Lanes 1 and 2). After transfection of pMV5 without the XER
expression plasmid, MV5 mRNA was observed at levels slightly lower than those seen in the presence of un-liganded estrogen receptor (see Fig. 2B; compare Lanes 3 and 4 with Lane 1). These data indicate that estradiol and estrogen receptor are both required to obtain the increased level of MV5 mRNA seen on estrogen stabiliza-tion.
To provide an internal standard that would also correct for any potential estrogen induction of transcription from the TK promoter, pMV5 and pTK7 were cotransfected into XL110 cells. As noted earlier, / h ~ 3 plasmid pTK7 contains the HSV TK structural gene driven by the HSV TK promoter. The levels of MV5 and TK mRNAs were simultaneously determined by the Sl nuclease pro-tection assay. The rQsults from three experiments in which the TK mRNA level was used as an internal standard to correct the level of MV5 mRNA confirm a requirement for estrogen receptor in the stabilization of MV5 mRNA
(see Table 2). Without estrogen receptor, estradiol was unable to stabilize MV5 mRNA. The data obtained in experiments using TK mRNA ae an internal transfection and assay standard and in the nine experiments summarized in Table 1, in which an internal standard was not used, are in excellent agreement, providing additional evidence that the increased level of MV5 mRNA in estrogen-treated cells is due to post-transcriptional mRNA stabilization rather than an increase in transcription from the TK
promoter.
As shown in Table 2, MN5 mRNA content in the absence of estrogen receptor was slightly lower than when estrogen receptor was present without added estradiol.
This result may have been due to partial stabilization resulting from traces of estrogen sulfate remaining in the charcoal-dextran-treated serum (~rown et al., Proc.
Natl. Acad. Sci. USA, 81, 6344-6348 (19841). The actual level of estrogen-dependent stabilization would then be somewhat greater than is presented in Tables 1 and 2.
Alternatively, the high level of unliganded estrogen receptor in pXER-tranafected cells may elicit partial stabilization of the M~5 mRNA. Since a TK internal standard was used in these experiments, even if this high level of unliganded receptor induced increased synthesis from the TK promoter, this effect would be corrected for in the data. Direct evidence that high levels of unliganded estrogen receptor do not induce increased expression of the TK promoter was obtained in additional experiments in which the TK-luciferase vector (see above) ~ ~ j h J S J W '~
was transiently cotransfected with either pXER or pTK-~-gal. Plasmid pTK-~-gal was added to maintain a constant TK promoter concentration when pXER was not used. The average levels of expression of the TK-luciferase vector in four separate experiments, performed similarly to those rsported in Table 2, differed by <1% in the presence or absence of pXER. This evidence and the use of the pTK7 internal standard in these experiments rule out the possibility that this result was due to increased transcription of the TK promoter induced by high levels of unliganded XER.
Since estrogen receptor does not destabilize MV5 mRNA in the absence of estradiol, the data exclude the possibility that MV5 mRNA is a stable mRNA that is destabilized by unliganded estrogen receptor. Maximum stabilization of MV5 mRNA occurred only when both the estrogen receptor and estradiol were present (Table 2), indicating that the estradiol-estrogen receptor complex plays a posi~ive role in stabilizing MV5 mRNA. This result is consistent with our observation that in the absence of estrogen, vitellogenin mRNA and total poly(A) mRNA exhibit the same half-life values. Brock and Shapiro, Cell, 34:207-214 (1983). These data support the hypothesis that in the absence of estrogen, vitellogenin mRNA is degraded by normal cellular mechanism. Blume et al., UCLA Symp. New Ser., 52, 259-274 (1987).
In pMV5, whose preparation is described in Example 1 above, there is a PstI site located at the junction of the two small vitellogenin coding sequences (see Figure lD). A DNA sequence coding for a protein or polypeptide of interest can be inserted into this PstI
site. DNA sequences coding for cleavage sites can also h ~`J ~ jJ
be inserted between the DNA sequence coding for the protein or polypeptide of interest and each of the vitellogenin coding regions in pMV5. Alternatively, the remaining small vitellogenin coding regions can be eliminated from pMV5 so that the DNA sequence coding for the protein or polypeptide of interest is linked directly to the DNA coding for the 3' and 5' untranslated regions.
Methods of preparing the DNA sequences and of inserting them into, or deleting them from, vectors such as pMV5 ara well-known.
The plasmid resulting from the above manipulations of pMV5 can be used to transform an approprlate ho~t (such as the XL110 cell line; see Example 2). If necessary, the host cells may also be transformed with a vector coding for estrogen receptor (see Example 2). When the transformed host cells are cultured in the presence of estrogen, the content of the hybrid mRNA coded for by the plasmid would increase substantially (up to 20-foldj as compared to the mRNA
content of cells maintained in the absence of estrogen.
As a consequence, enhanced production of the protein or polypeptide of interest coded for by the hybrid mRNA
would be obtained.
Plasmid pTK-~-gal is a TK promoter-~-galactosidase gene fusion plasmid. It was constructed by inserting the TK promoter in place of the SV40 promoter in pCHllO. This was accomplished by digesting pCHllO
with KpnI and PvuII and blunt ending the sites by mung bean digestion. The TK promoter was excised ~rom pXER by digestion with EcoRI and PstI. The ends were made blunt by mung bean digestion, and the fragments were joined by blunt end ligation. Plasmid pCHllO is a SV40-~-galactosidase fusion plasmid available from Pharmacia.
`
D. E~ isolation RNA was isolated from transfected XL110 cells at the indicated times after glycerol shock by the guanidine thiocyanate-phenol-chloroform method which is described in Chromczynski and Sacchi, Anal. Biochem., 162, 156-159 (1987). ~enopus liver RNA, isolated from uninduced livers and from livers induced for 12 days with estradiol, was isolated as previoualy described in Shapiro and Bak~r, J. Biol. Chem., 252, 5244-5250 (1977).
E. Solution hybridization and S1 nuclease protection assay Solution hybridization and the S1 nuclease protection assay were performed as descrîbed previously in Salton et al., Mol. Endocrinol., 2, 1033-1042 (1988), with the following modifications. A 2-ng sample of 32p_ labeled cRNA probe (2.7 x 106 cpm) was hybridized to the indicated amount of RNA at 50C overnight. S1 nuclease (1,000 U; Pharmacia, Inc.) was used for digestion at 43C
for 90 min.
After S1 nuclease digestion, samples were electrophoresed in a 4% polyacrylamide gel, using the 1-kb DNA ladder (Bethesda Research Laboratories) as a molecular size standard. Autoradiograms were scanned by using an LKB UltroScan XL densitometer.
Figure 3 presents a schematic of the probes used in the S1 nuclease protection assays and the sizes of the parts of the probes protected by MV5, vitellogenin B1, and TK m~NAs. The 1036-nucleotide MV5 RNA probe used to detect MV5 mRNA in the S1 nuclease protection assay is also shown in Figure lA above the pMV5 construction. The probes were synthesized as described in Salton et al., Mol. Endoc~inol., 2, 1033-42 (1988).
~?.~
F. Results The results of the transfections and the Sl nuclease as~ays are shown in Figures 2A-B and in Tables 1 and 2~ In Figure 2A, the results are shown of an assay in which four plates of XL110 cells were each transfected with 20 ~g of pMV5 and 20 ~g of pXER. RNA isolated from these cell~ was analyzed by the S1 nuclease protection assay using only the MV5 cRNA probe (see Figures lA and 3).
In Figure 2A, the lanes contained the following materials: Lanes 1 and 2---30 ~g of RNA isolated from transfected XLllO cells grown in the absence (Lane 1) or presence (Lane 2) of 100 nM estradiol-17~; Lane 3---10 ~g of RNA from uninduced male Xenopus liver; Lane 4---1 ng of RNA from Xenopus liver induced for 12 days with estrogen plus 10 ~g of uninduced Xenopus liver RNA.
In Figure 2B, the RNA analyzed was from cells transfected as follows: Lanes 1 and 2---10 ~g each of pMV5, pXER, and pTK7; Lanes 3 and 4---10 ~g each of pMV5, pTX7, and pTKCAT; Lane 5---50 ng of HSV-infected Vero cell RNA (obtained from Bernard Roizman, The University of Chicago Medical School, Chicago, Illinois) plus 10 ~g of uninduced Xenopus liver RNA; Lane 6---1 ng of RNA from a Xeno~us liver induced for 12 days with estradiol plus 10 ~g of uninduced Xenopus RNA; Lane 7, 10 ~g of RNA from uninduced male Xeno~us liver. The RNA electrophoresed in Lanes 1 and 3 was from cultures grown for 72 hours in the absence of hormone, and that in Lanes 2 and 4 from cultures grown for 72 hours in 100 nM estradiol-17~. The autoradiograms for Lanes 1, 2, 5, 6, and 7 were exposed for 3 days; those for Lanes 3 and 4 were exposed for 22 hours.
Table 1 presents the MV5 mRNA content found in transfected XLllO cells cultured in the presence of 100 nM estradiol-17~ relative to the MV5 mRNA content in cells cultured in the absence of hormone. The MV5 mRNA
~ ~ 2,, ~ 1 ~
content is expressed as mean + standard error of the mean, and the figures given are the means of nine independent transfection experiments. In each experi-ment, X~110 cells were transfected with 10 or 20 ~g of pMV5 and an equal amount of pXER. RNA isolated from the transfected cells was analyzed by the Sl nuclease pro-tection assay u6ing only the MV5 cRNA probe (see Figures lA and 3).
Control of MV5 mRNA content by estradiol Relative content of MV5 mRNA
Culture Condition (mean + SEM) No hormone 100 100 nN estradiol 164 + 13 Shown in Table 2 are the results of co-transfecting XLllO cells with 10 ~g each pMV5, pTK7 and pXER or with 10 ~g each pMV5, pTK7 and pTKCAT. After 72 hour~, RNA was isolated from the transfected cells, and the levels of MV5 and TK mRNAs were simultaneously determined by the S1 nuclease protection assay. The ratio of MV5 mRNA content to TK mRNA content was used to calculate the content of MV5 mRNA relative to that in cells transfected with pMV5, pXER and pTK7 and grown in no hormone. Plasmid pTK7 was used to provide an internal Rtandard that would correct for any potential estrogen induction of transcription from the TK promoter. When ~ 2 ~Vi w ~ ~ ~
pXER was not used, plasmid pTKCAT was used to keep the level of TK promoter the same in all cells.
Control of MV5 mRNA content by estradiol and estrogen receptor Relative content of MV5 mRNA
DNA~ Culture Condition ~mean ~ SEM) pMV5, pXER No hormone 100 pMV5, pXER 100 nM estradiol 163 + 19 p~V5, pTKCAT No hormone 62 + 12 pMV5, pTKCAT 100 nM estradiol 72 + 12 aAll cells were also transfected with pTK7.
G. Discussion of results 1. Production of MV5 mRNA
Cotransfection of pMV5 and pXER into Xenopus XL110 liver cells resulted in the production of MV5 mRNA.
A 534-nucleotide region of the MV5 cRNA probe, corresponding to the 529-nucleotide MV5 mRNA and the first 5 nucleotides of the poly(A) tail, was protected in the S1 nuclease assay (see Fig. 2A and 2B). Uninduced Xeno~us liver RNA (see Fig. 2A, lane 3; Fig. 2B, lane 7) and mock-transfected XL110 cell RNA (data not shown) produced no protected bands in this size range. Vitel-logenin mRNA from estradiol-induced Xenopus liver pro-tected a 440-nucleotide band, corresponding to the 3' 435 nucleotides of vitellogenin mRNA and the first 5 2 ~ . ~o~ ~i c ~ . ~
nucleotides of the poly(A) tail (see Fig. 2A, lane 4;
Fig. 2B, lane 6). The small 90-nucleotide fragment corresponding to the region of the probe protected by the 5' end o~ full-length vitellogenin mRNA was not seen because it was run off the gel.
2. Role of estrogen in stabilization of MV5 mRNA
To test whether the MV5 mRNA could be stabil-ized by estrogen, pMV5 and pXER were transfected into XLllO cells, and the cells were maintained for 72 hours in the presence or absence of estrogen. MV5 m~NA was stabilized by eRtrogen (see Fig. 2A, Lanes 1 and 2).
When the data from nine separate experiments were aver-aged, the MV5 mRNA content of cells maintained in estradiol was increased by 64 + 13% (see Table 1) com-pared to cells grown in the absence of hormone.
The relative level of MV5 mRNA expected after estrogen stabilization can be calculated by using data obtained from studies of the stability of full-length vitellogenin mRNA in Xenopus liver fragment cultures (Brock and Shapiro, Cell, 34, 207-214 (1983); Blume and Shapiro, Nucleic Acids Res., 17, 9003-9014 (1989)) and from transfection data on the accumulation of MV5 mRNA
and chloramphenicol acetyltransferase enzymatic activity at various times after transfection. Those studies indicate that at 20C there is no significant transcription for 12 hours after transfection into XLllo cells and that transcription for the next 12 hours is at a reduced rate (data not shown). Assuming that transcription then occurs at a constant rate between 24 and 72 hours post-transfection an~ that the MV5 mRNA
exhibits a half-life of 30 hours at 20C in the absence of estrogen and 500 hours (infinite in this calculation) in the presence of estrogen, then we would expect a 1.68-fold increase in MV5 mRNA content due to stabilization by ~J V 2 ~ ~.
estrogen 72 hours after transfection. This value is in excellent agreement with the average 1.64-fold increase in MV5 mRNA levels that was observed (see Table 1).
Because of the relatively long half-life of vitellogenin mRNA in the absence of estrogen, even if transcription of the MV5 gene occurred at a constant rate throughout the 72 hours, estrogen stabilization would result in only a 1.9-fold increa~e in NV5 mRNA content.
Although these data provide strong support for the view that the estradiol-estrogen receptor complex regulates the stability of the MV5 mRNA, they do not explicitly exclude the possibility that this complex induce~ increased transcription of the MV5 gene. Direct evidence that transcription of the TK promoter used in the pMV5 vector is not regulated by estrogen was obtained from experiments in which the same TK promoter in a TK-luciferase vector and plasmid pXER were cotransfected into XL110 cells. In 16 transient cotransfection experiments carried out under conditions similar to those described above, the average level of expression of the TK-luciferase plasmid was 665 luminescence units per ~g in the absence of estradiol and 670 luminescence units per ~g in the presence of estradiol. These values differ by <1%. This result provides compelling evidence that expression of the TK promoter is not induced by the estradiol-XER complex.
The related possibility that the vitellogenin DNA sequence cloned into the pMV5 vector contains an estrogen response element (ERE) that functions as a 3' enhancer is excluded by examination of the sequence of pMV5. The entire nucleotide sequence of the MV5 DNA and the 3'-flanking genomic fragment inserted into the pMV5 clone has been determined. There is no sequence either identical to the consensus ERE or diverging from the ERE
by one, two, or even three nucleotides. All known functional EREs contain zero to two nucleotide changes , ~J ~ ~F"~J 3 c ~ ~
relative to the consensus ERE, indicating that there is no functional estrogen receptor-binding site in the MV5 DNA and its 3'-flanking sequence.
Therefore, three lines of evidence support the conclusion that the estradiol-estrogen receptor complex stabilized MV5 mRNA: (1) the failure of the estradiol-Qstrog~n receptor complex to induce transcription from the TK promoter used in the pMV5 plasmid; (2) the absence of any sequence related to the ERE with the potential to serv2 a~ a 3' transcription enhancer and estrogen receptor-binding site in the MV5 DNA and its flanking DNA; and (3) the excellent agreement between the observed level of MV5 mRNA and the predicted level for an mRNA
undergoing estrogen-mediated stabilization.
3. Requirement for estrogen recetor for MV5 mRNA stabilization In the experiments described above, high levels of estrogen receptor were synthesized from the transfected pXER plasmid. To determine whether the estrogen receptor is required for stabilization of MV5 mRNA, cells were transfected with pMV5 and pXER or with pMV5 and pTKCAT. When pMV5 and pXER were cotransfected, MV5 mRNA was stabilized by estrogen (see Fig. 2B, Lanes 1 and 2). After transfection of pMV5 without the XER
expression plasmid, MV5 mRNA was observed at levels slightly lower than those seen in the presence of un-liganded estrogen receptor (see Fig. 2B; compare Lanes 3 and 4 with Lane 1). These data indicate that estradiol and estrogen receptor are both required to obtain the increased level of MV5 mRNA seen on estrogen stabiliza-tion.
To provide an internal standard that would also correct for any potential estrogen induction of transcription from the TK promoter, pMV5 and pTK7 were cotransfected into XL110 cells. As noted earlier, / h ~ 3 plasmid pTK7 contains the HSV TK structural gene driven by the HSV TK promoter. The levels of MV5 and TK mRNAs were simultaneously determined by the Sl nuclease pro-tection assay. The rQsults from three experiments in which the TK mRNA level was used as an internal standard to correct the level of MV5 mRNA confirm a requirement for estrogen receptor in the stabilization of MV5 mRNA
(see Table 2). Without estrogen receptor, estradiol was unable to stabilize MV5 mRNA. The data obtained in experiments using TK mRNA ae an internal transfection and assay standard and in the nine experiments summarized in Table 1, in which an internal standard was not used, are in excellent agreement, providing additional evidence that the increased level of MV5 mRNA in estrogen-treated cells is due to post-transcriptional mRNA stabilization rather than an increase in transcription from the TK
promoter.
As shown in Table 2, MN5 mRNA content in the absence of estrogen receptor was slightly lower than when estrogen receptor was present without added estradiol.
This result may have been due to partial stabilization resulting from traces of estrogen sulfate remaining in the charcoal-dextran-treated serum (~rown et al., Proc.
Natl. Acad. Sci. USA, 81, 6344-6348 (19841). The actual level of estrogen-dependent stabilization would then be somewhat greater than is presented in Tables 1 and 2.
Alternatively, the high level of unliganded estrogen receptor in pXER-tranafected cells may elicit partial stabilization of the M~5 mRNA. Since a TK internal standard was used in these experiments, even if this high level of unliganded receptor induced increased synthesis from the TK promoter, this effect would be corrected for in the data. Direct evidence that high levels of unliganded estrogen receptor do not induce increased expression of the TK promoter was obtained in additional experiments in which the TK-luciferase vector (see above) ~ ~ j h J S J W '~
was transiently cotransfected with either pXER or pTK-~-gal. Plasmid pTK-~-gal was added to maintain a constant TK promoter concentration when pXER was not used. The average levels of expression of the TK-luciferase vector in four separate experiments, performed similarly to those rsported in Table 2, differed by <1% in the presence or absence of pXER. This evidence and the use of the pTK7 internal standard in these experiments rule out the possibility that this result was due to increased transcription of the TK promoter induced by high levels of unliganded XER.
Since estrogen receptor does not destabilize MV5 mRNA in the absence of estradiol, the data exclude the possibility that MV5 mRNA is a stable mRNA that is destabilized by unliganded estrogen receptor. Maximum stabilization of MV5 mRNA occurred only when both the estrogen receptor and estradiol were present (Table 2), indicating that the estradiol-estrogen receptor complex plays a posi~ive role in stabilizing MV5 mRNA. This result is consistent with our observation that in the absence of estrogen, vitellogenin mRNA and total poly(A) mRNA exhibit the same half-life values. Brock and Shapiro, Cell, 34:207-214 (1983). These data support the hypothesis that in the absence of estrogen, vitellogenin mRNA is degraded by normal cellular mechanism. Blume et al., UCLA Symp. New Ser., 52, 259-274 (1987).
In pMV5, whose preparation is described in Example 1 above, there is a PstI site located at the junction of the two small vitellogenin coding sequences (see Figure lD). A DNA sequence coding for a protein or polypeptide of interest can be inserted into this PstI
site. DNA sequences coding for cleavage sites can also h ~`J ~ jJ
be inserted between the DNA sequence coding for the protein or polypeptide of interest and each of the vitellogenin coding regions in pMV5. Alternatively, the remaining small vitellogenin coding regions can be eliminated from pMV5 so that the DNA sequence coding for the protein or polypeptide of interest is linked directly to the DNA coding for the 3' and 5' untranslated regions.
Methods of preparing the DNA sequences and of inserting them into, or deleting them from, vectors such as pMV5 ara well-known.
The plasmid resulting from the above manipulations of pMV5 can be used to transform an approprlate ho~t (such as the XL110 cell line; see Example 2). If necessary, the host cells may also be transformed with a vector coding for estrogen receptor (see Example 2). When the transformed host cells are cultured in the presence of estrogen, the content of the hybrid mRNA coded for by the plasmid would increase substantially (up to 20-foldj as compared to the mRNA
content of cells maintained in the absence of estrogen.
As a consequence, enhanced production of the protein or polypeptide of interest coded for by the hybrid mRNA
would be obtained.
Claims (9)
1. A hybrid mRNA molecule comprising a first RNA
sequence coding for a protein or polypeptide operatively linked to a second RNA sequence which is capable of stabilizing the hybrid mRNA in the presence of estrogen and estrogen receptor.
sequence coding for a protein or polypeptide operatively linked to a second RNA sequence which is capable of stabilizing the hybrid mRNA in the presence of estrogen and estrogen receptor.
2. The hybrid mRNA molecule of Claim 1 wherein the second RNA sequence comprises a portion of a vitellogenin mRNA.
3. The hybrid mRNA molecule of Claim 2 wherein the second RNA sequence comprises two parts, the first part, which is located 5' of the first RNA sequence, comprises the 5' untranslated region of the vitellogenin B1 mRNA linked to 81 nucleotides coding for the amino-terminal 27 amino acids of vitel-logenin B1, and the second part, which is located 3' of the first RNA sequence, comprises 270 nucleotides coding for the carboxy-terminal 90 amino acids of vitellogenin B1 linked to the 3' untranslated region of the vitellogenin B1 mRNA.
4. The hybrid mRNA molecule of Claim 1 further comprising a third sequence located between, and oper-atively linked to, the first and second sequences, the third sequence coding for a cleavage site.
5. The hybrid mRNA molecule of Claim 3 further comprising third and fourth sequences, both coding for cleavage sites, the third sequence being located between, and operatively linked to, the first sequence and the first part of the second sequence, and the fourth sequence being located between, and operatively linked to, the first sequence and the second part of the second sequence.
6. A recombinant DNA molecule coding for the hybrid mRNA of Claim 1, 2, 3, 4 or 5.
7. A vector comprising the recombinant DNA mole-cule of Claim 6 operatively linked to expression control sequences.
8. A host cell transformed with the vector of Claim 7.
9. A method of stabilizing an mRNA so as to obtain increased production of a protein or polypeptide comprising:
transforming a host cell with the vector of Claim 7;
also transforming the host cell with a vector coding for estrogen receptor if the host cell does not normally produce estrogen receptor; and growing the transformed host cell in the presence of estrogen.
transforming a host cell with the vector of Claim 7;
also transforming the host cell with a vector coding for estrogen receptor if the host cell does not normally produce estrogen receptor; and growing the transformed host cell in the presence of estrogen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2022356 CA2022356A1 (en) | 1990-07-31 | 1990-07-31 | Method and materials for producing proteins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2022356 CA2022356A1 (en) | 1990-07-31 | 1990-07-31 | Method and materials for producing proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2022356A1 true CA2022356A1 (en) | 1992-02-01 |
Family
ID=4145601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2022356 Abandoned CA2022356A1 (en) | 1990-07-31 | 1990-07-31 | Method and materials for producing proteins |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2022356A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000026366A1 (en) * | 1998-10-30 | 2000-05-11 | National University Of Singapore | Isolated nucleic acids encoding a secretory signal for expression and secretion of heterologous recombinant proteins |
| WO2014113820A1 (en) * | 2013-01-18 | 2014-07-24 | Shapiro David J | Estrogen receptor inhibitors |
-
1990
- 1990-07-31 CA CA 2022356 patent/CA2022356A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000026366A1 (en) * | 1998-10-30 | 2000-05-11 | National University Of Singapore | Isolated nucleic acids encoding a secretory signal for expression and secretion of heterologous recombinant proteins |
| WO2014113820A1 (en) * | 2013-01-18 | 2014-07-24 | Shapiro David J | Estrogen receptor inhibitors |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0117058B1 (en) | Methods for producing mature protein in vertebrate host cells | |
| CA2009306C (en) | Expression systems for amidating enzyme | |
| Huang et al. | The simian virus 40 small-t intron, present in many common expression vectors, leads to aberrant splicing | |
| US4840896A (en) | Heteropolymeric protein | |
| EP0173552B1 (en) | Recombinant dna compounds and the expression of polypeptides such as tpa | |
| AU692196B2 (en) | Translational enhancer DNA | |
| KR100257457B1 (en) | Method for preparing human coagulation factor VIII protein complex | |
| Dreano et al. | High-level, heat-regulated synthesis of proteins in eukaryotic cells | |
| EP0160699B1 (en) | Production of heterodimeric human fertlity hormones | |
| Widen et al. | Liver-specific expression of the mouse alpha-fetoprotein gene is mediated by cis-acting DNA elements. | |
| NO179106B (en) | Method of Preparation and Expression System Encoding and Expression of a Recombinant Protein Complex with Human Factor V111: C Activity | |
| US4965196A (en) | Polycistronic expression vector construction | |
| CA2148492C (en) | Bovine heat shock promoter and uses thereof | |
| Nikovits Jr et al. | Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon | |
| US4939088A (en) | Sustained production of recombinant gamma interferon using an Epstein-Barr virus replicon | |
| CA2022356A1 (en) | Method and materials for producing proteins | |
| Kato et al. | Isolation and functional expression of pituitary peptidylglycine α‐amidating enzyme mRNA: A variant lacking the transmembrane domain | |
| AU604214B2 (en) | An improved heat-shock control method and system for the production of competent eukaryotic gene products | |
| JPH1052266A (en) | Production of tissue plasminogen activation factor | |
| CA1224168A (en) | Human growth hormone produced by recombinant dna in mouse cells | |
| Smith et al. | Proximal 2.6 kb of 5′-flanking DNA is insufficient for human renin promoter activity in renin-synthesizing chorio-decidual cells | |
| WO1987005935A1 (en) | Methods and compositions for expression of competent eukaryotic gene products | |
| WO1992002529A1 (en) | Expression vectors regulated by estrogen and a mutant estrogen receptor | |
| US5646010A (en) | Methods and compositions for expression of competent eukaryotic gene products | |
| Doyen et al. | Analysis of promoter and enhancer cell type specificities and the regulation of immunoglobulin gene expression |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |