Post transcriptional gene regulation by selenium
First Claim
1. A method of controlling the production in a eukaryotic cell of a heterologous polypeptide that does not contain selenocysteine in its native form, said method comprising,(1) transfecting a cell with (i) a first nucleic acid encoding said heterologous polypeptide, wherein at least one codon of mRNA transcribed from said first nucleic acid is replaced by the codon UGA, and (ii) a second nucleic acid operably linked to said first nucleic acid, said second nucleic acid directing the translation of said UGA codon as selenocysteine only when said cell can obtain selenium from the medium in which said cell is grown, wherein said second nucleic acid comprises a continuous stretch of at least 79 nucleotides comprising three stem elements, each having a 5'"'"' half and a 3'"'"' half, and three loop elements, each having a 5'"'"' end and a 3'"'"' end, wherein the stem elements comprisea) a base stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides,b) a lower stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides, the first nucleotide of the 5'"'"' half of the lower stem being bound to the last nucleotide of the 5'"'"' half of the base stem, and the first nucleotide of the 3'"'"' half of the lower stem being bound to the last nucleotide of the 3'"'"' half of the base stem, andc) an upper stem comprising at least 22 nucleotides that can form 11 complementary pairs of nucleotides,wherein the loop elements comprised) a first loop consisting of 5'"'"'-AUGRG-3'"'"' (SEQ ID NO:
- 26), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the lower stem and the 3'"'"'-G being bound to the first nucleotide of the 5'"'"' half of the upper stem,e) a second loop consisting of 5'"'"'-YRNNNNUAV-3'"'"' (SEQ ID NO;
27), the 5'"'"'-Y being bound to the first nucleotide of the 3'"'"' half of the upper stem and the 3'"'"'-V being bound to the last nucleotide of the 3'"'"' half of the lower stem, andf) a third, apical loop consisting of 5'"'"'-ARANNNNNNNN-3'"'"' (SEQ ID NO;
28), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the upper stem and the 3'"'"'-N being bound to the last nucleotide of the 3'"'"' half of the upper stem, andwherein each A is adenine, G is guanine, N is adenine, guanine, cytosine, or uracil, R is guanine or adenine, U is uracil, V is any nucleotide except thymidine or uracil, and Y is uracil or cytosine; and
(2) growing said cell in culture under conditions wherein the production of said polypeptide is controlled by the level of selenium available to said cell.
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Accused Products
Abstract
Methods of controlling the in vivo and in vitro expression of a heterologous protein by transfecting a cell with a first nucleic acid encoding the heterologous polypeptide, wherein at least one codon of mRNA transcribed from the first nucleic acid is replaced by the codon UGA, and a second nucleic acid operably linked to the first nucleic acid, the second nucleic acid directing the translation of the UGA codon as selenocysteine only when the cell can obtain selenium from the medium in which it is grown; and growing the cell under conditions in which the production of the polypeptide is controlled by the level of selenium available to the cell.
15 Citations
16 Claims
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1. A method of controlling the production in a eukaryotic cell of a heterologous polypeptide that does not contain selenocysteine in its native form, said method comprising,
(1) transfecting a cell with (i) a first nucleic acid encoding said heterologous polypeptide, wherein at least one codon of mRNA transcribed from said first nucleic acid is replaced by the codon UGA, and (ii) a second nucleic acid operably linked to said first nucleic acid, said second nucleic acid directing the translation of said UGA codon as selenocysteine only when said cell can obtain selenium from the medium in which said cell is grown, wherein said second nucleic acid comprises a continuous stretch of at least 79 nucleotides comprising three stem elements, each having a 5'"'"' half and a 3'"'"' half, and three loop elements, each having a 5'"'"' end and a 3'"'"' end, wherein the stem elements comprise a) a base stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides, b) a lower stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides, the first nucleotide of the 5'"'"' half of the lower stem being bound to the last nucleotide of the 5'"'"' half of the base stem, and the first nucleotide of the 3'"'"' half of the lower stem being bound to the last nucleotide of the 3'"'"' half of the base stem, and c) an upper stem comprising at least 22 nucleotides that can form 11 complementary pairs of nucleotides, wherein the loop elements comprise d) a first loop consisting of 5'"'"'-AUGRG-3'"'"' (SEQ ID NO: - 26), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the lower stem and the 3'"'"'-G being bound to the first nucleotide of the 5'"'"' half of the upper stem,
e) a second loop consisting of 5'"'"'-YRNNNNUAV-3'"'"' (SEQ ID NO;
27), the 5'"'"'-Y being bound to the first nucleotide of the 3'"'"' half of the upper stem and the 3'"'"'-V being bound to the last nucleotide of the 3'"'"' half of the lower stem, andf) a third, apical loop consisting of 5'"'"'-ARANNNNNNNN-3'"'"' (SEQ ID NO;
28), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the upper stem and the 3'"'"'-N being bound to the last nucleotide of the 3'"'"' half of the upper stem, andwherein each A is adenine, G is guanine, N is adenine, guanine, cytosine, or uracil, R is guanine or adenine, U is uracil, V is any nucleotide except thymidine or uracil, and Y is uracil or cytosine; and (2) growing said cell in culture under conditions wherein the production of said polypeptide is controlled by the level of selenium available to said cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
- 26), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the lower stem and the 3'"'"'-G being bound to the first nucleotide of the 5'"'"' half of the upper stem,
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12. A synthetic single-stranded nucleic acid capable of forming a stem-loop secondary structure, comprising a continuous stretch of at least 79 nucleotides comprising three stem elements, each having a 5'"'"' half and a 3'"'"' half, and three loop elements, each having a 5'"'"' end and a 3'"'"' end,
wherein the stem elements comprise a) a base stem comprising at least 16 nucleotides which form 8 complementary pairs of nucleotides, b) a lower stem comprising at least 16 nucleotides which form 8 complementary pairs of nucleotides, the first nucleotide of the 5'"'"' half of the lower stem being bound to the last nucleotide of the 5'"'"' half of the base stem, and the first nucleotide of the 3'"'"' half of the lower stem being bound to the last nucleotide of the 3'"'"' half of the base stem, and c) an upper stem comprising at least 22 nucleotides which form 11 complementary pairs of nucleotides, wherein the loop elements comprise d) a first loop consisting of 5'"'"'-AUGRG-3'"'"' (SEQ ID NO: - 26), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the lower stem and the 3'"'"'-G being bound to the first nucleotide of the 5'"'"' half of the upper stem,
e) a second loop consisting of 5'"'"'-YRNNNNUAV-3'"'"' (SEQ ID NO;
27), the 5'"'"'-Y being bound to the first nucleotide of the 3'"'"' half of the upper stem and the 3'"'"'-V being bound to the last nucleotide of the 3'"'"' half of the lower stem, andf) a third, apical loop consisting of 5'"'"'-ARANNNNNNNN-3'"'"' (SEQ ID NO;
28), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the upper stem and the 3'"'"'-N being bound to the last nucleotide of the 3'"'"' half of the upper stem, andwherein each A is adenine, G is guanine, N is adenine, guanine, cytosine, or uracil, R is guanine or adenine, U is uracil, V is any nucleotide except thymidine or uracil, and Y is uracil or cytosine. - View Dependent Claims (13, 14)
- 26), the 5'"'"'-A being bound to the last nucleotide of the 5'"'"' half of the lower stem and the 3'"'"'-G being bound to the first nucleotide of the 5'"'"' half of the upper stem,
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15. An isolated nucleic acid having the sequence of FIG. 9E (SEQ ID NO:
- 1).
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16. An isolated nucleic acid having the sequence of FIG. 9F (SEQ ID NO:
- 2).
Specification