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AssigneesCubist Pharmaceuticals, Inc., Cancer Institute, Japanese Foundation for Cancer Research, Massachusetts Institute of Technology
InventorsKiyotaka Shiba, Paul R. Schimmel, Tracy L. Ripmaster
Estimating the term…
Isolated, recombinant nucleic acids which encode alanyl-tRNA synthetase (AlaRS) of human origin have been used to make expression constructs and transformed host cells for the production of recombinant human AlaRS. The recombinant enzyme has been purified, and is active in the specific aminoacylation of tRNA by alanine. The isolated, recombinant human AlaRS is also recognized by antibodies made by patients with the particular autoimmune disease known as "antisynthetase syndrome" in which the patients produce antibodies against the human alanyl-tRNA synthetase in their own cells. Thus, the isolated, recombinant enzyme, and antibodies made specifically thereto, can be useful in assays to diagnose and monitor this disease. The essential alanyl-tRNA synthetases of microbes pathogenic in humans can be the targets of inhibitory agents having antimicrobial activity. The human alanyl-tRNA synthetase, isolated and purified, can be used to assess the toxic effect in humans of such an inhibitory agent in various biochemical activity assays. This human enzyme can also be expressed in "tester strains," whose cells rely upon the function of the human alanyl-tRNA synthetase for tRNA.sup.Ala charging. Such tester strains can be used to test for any toxic effects of an antimicrobial agent that specifically interacts with the heterologous human AlaRS gene or gene product.
An essentially pure nucleic acid which encodes a human alanyl-tRNA synthetase.
An essentially pure nucleic acid which codes for an active human alanyl-tRNA synthetase, and which hybridizes to DNA having SEQ ID NO:1 under moderate stringency conditions, wherein moderate stringency conditions comprise hybridization in 6X SSC, 1% sodium dodecyl sulfate, 20 mM NaH.sub.2 PO.sub.4 and 500 .mu.g/ml salmon sperm DNA at 42.degree. C. for 16 hours and two washes in 6X SSC and 0.1% sodium dodecyl sulfate at 56.degree. C. for 15 minutes.
An essentially pure nucleic acid which encodes an amino acid sequence SEQ ID NO:2.
An isolated nucleic acid comprising a nucleic acid having a sequence complementary to a DNA strand having a SEQ ID NO:1 or to an RNA counterpart of SEQ ID NO:1 or to a portion of said DNA or RNA counterpart comprising the coding sequence.
A recombinant nucleic acid vector comprising nucleic acid which encodes a human alanyl-tRNA synthetase and which hybridizes to DNA having SEQ ID NO:1 under moderate stringency conditions, wherein moderate stringency conditions comprise hybridization in 6X SSC, 1% sodium dodecyl sulfate, 20 mM NaH.sub.2 PO.sub.4 and 500 .mu.g/ml salmon sperm DNA at 42.degree. C. for 16 hours and two washes in 6X SSC and 0.1% sodium dodecyl sulfate at 56.degree. C. for 15 minutes.
A recombinant nucleic acid vector comprising DNA which encodes a human alanyl-tRNA synthetase.
An expression vector comprising a nucleic acid encoding a fusion protein comprising a human alanyl-tRNA synthetase, wherein said nucleic acid comprises a coding sequence for a human alanyl-tRNA synthetase, and wherein the coding sequence is under control of transcription signals and is linked to appropriate translation signals for expression in a suitable host cell.
A host cell comprising a recombinant human alanyl-tRNA synthetase gene.
A method for producing active human alanyl-tRNA synthetase comprising the following steps:
A method for producing isolated, recombinant human alanyl-tRNA synthetase comprising the following steps:
A host cell comprising a recombinant nucleic acid encoding a fusion protein comprising a human alanyl-tRNA synthetase.
A tester strain comprising host cells which comprise a recombinant human alanyl-tRNA synthetase gene which expresses a human alanyl-tRNA synthetase, wherein the recombinant, human alanyl-tRNA synthetase gene complements or substitutes in function for a host cell alanyl-tRNA synthetase gene.
An essentially pure nucleic acid which encodes a Saccharomyces cerevisiae alanyl-tRNA synthetase and which hybridizes to DNA having SEQ ID NO:3 under moderate stringency conditions, wherein moderate stringency conditions comprise hybridization in 6X SSC. 1% sodium dodecyl sulfate, 20 mM NaH.sub.2 PO.sub.4 and 500 .mu.g/ml salmon sperm pNA at 42.degree. C. for 16 hours and two washes in 6X SSC and 0.1% sodium dodecyl sulfate at 56.degree. C. for 15 minutes.
An essentially pure nucleic acid which encodes an alanyl-tRNA synthetase of Saccharomyces cerevisiae having the amino acid sequence in SEQ ID NO:4.
A recombinant nucleic acid vector comprising nucleic acid which encodes alanyl-tRNA synthetase of Saccharomyces cerevisiae.
A host cell comprising a recombinant Saccharomyces cerevisiae alanyl-tRNA synthetase gene which expresses a Saccharomyces cerevisiae alanyl-tRNA synthetase or fusion protein which comprises a Saccharomyces cerevisiae alanyl-tRNA synthetase.
A host cell comprising a recombinant nucleic acid which encodes a human alanyl-tRNA synthetase and which hybridizes to DNA having SEQ ID NO:1 under moderate stringency conditions, wherein moderate stringency conditions comprise hybridization in 6X SSC, 1% sodium dodecyl sulfate, 20 mM NaH.sub.2 PO.sub.4 and 500 .mu.g/ml salmon sperm DNA at 42.degree. C. for 16 hours and two washes in 6X SSC and 0.1% sodium dodecyl sulfate at 56.degree. C. for 15 minutes.
An isolated nucleic acid, wherein said nucleic acid encodes a protein comprising an alanyl-tRNA synthetase which is encoded by pKS-AA1-12 as deposited under ATCC Accession No. 98270.
A host cell comprising a recombinant nucleic acid, wherein said nucleic acid encodes a protein comprising an alanyl-tRNA synthetase which is encoded by pKS-AA1-12 as deposited under ATCC Accession No. 98270.
Plasmid pKS-AA1-12, from host cells deposited under ATCC Accession No. 98270.
An isolated nucleic acid, Wherein said nucleic acid encodes a protein comprising an alanyl-tRNA synthetase which is encoded by pTR94 as deposited under ATCC Accession No. 98269.
A recombinant nucleic acid vector comprising nucleic acid encoding a protein comprising an alanyl-tRNA synthetase which is encoded by pTR94 as deposited under ATCC Accession No. 98269.
A host cell comprising a recombinant nucleic acid, wherein said nucleic acid encodes a protein comprising an alanyl-tRNA synthetase which is encoded by pTR94 as deposited under ATCC Accession No. 98269.
Plasmid pTR94, from host cells deposited under ATCC Accession No. 98269 .
Dependencies are inferred from the claim text.