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Claims for Patent: 6,365,127

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Claims for Patent: 6,365,127

Title: Isolation of a gene encoding human thyrotropin beta subunit
Abstract:The gene expressing the beta subunit of human thyroid stimulating hormone has been isolated. The gene has been incorporated into plasmid pBR322. Vectors can be used to transform cells which in turn produce pure beta subunits. The beta subunits can then be combined with the alpha subunit of human glycoprotein hormones to produce pure thyroid stimulating hormone.
Inventor(s): Kourides; Ione A. (Forest Hills, NY), Whitfield; Graham Kerr (New York, NY)
Assignee: Sloan-Kettering Institute for Cancer Research (New York, NY)
Application Number:09/516,352
Patent Claims: 1. A thyroid stimulating hormone beta chain produced by a cell transformed with a vector comprising a DNA sequence encoding the beta subunit of human thyroid stimulating hormone, wherein the DNA sequence comprises the following sequence:

(a) a first sequence ATG ACT GCT CTC TTT CTG ATG TCC ATG CTT TTT GGC CTT GCA TGT GGG CAA GCG ATG TCT TTT TGT ATT CCA ACT GAG TAT ACA ATG CAC ATC GAA AGG AGA GAG TGT GCT TAT TGC CTA ACC ATC AAC ACC ACC ATC TGT GCT GGA TAT TGT ATG ACA CGG;

(b) a second sequence of about 400-450 nucleotides following the first sequence; and

(c) a third sequence GAT ATC AAT GGC AAA CTG TTT CTT CCC AAA TAT GCT CTG TCC CAG GAT GTT TGC ACA TAT AGA GAC TTC ATC TAC AGG ACT GTA GAA ATA CCA GGA TGC CCA CTC CAT GTT GCT CCC TAT TTT TCC TAT CCT GTT GCT TTA AGC TGT AAG TGT GGC AAG TGC AAT ACT GAC TAT AGT GAC TGC ATA CAT GAA GCC ATC AAG ACA AAC TAC TGT ACC AAA CCT CAG AAG TCT TAT CTG GTA GGA TTT TCT GTC TAA following the second sequence.

2. A method of determining the cause of a thyroid gland failure in a subject comprising: administering to the subject human thyroid stimulating hormone prepared by recombinant DNA technology; and determining whether such administration results in a change in the activity of said subject's thyroid gland, a change indicating that the thyroid gland failure results from central pituitary disease, and no change indicating primary thyroid failure.

3. Substantially pure human thyroid stimulating hormone comprising a beta subunit prepared by recombinant DNA technology and having the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

4. Substantially pure human thyroid stimulating hormone comprising a beta subunit produced using a plasmid comprising a DNA sequence encoding said subunit, which plasmid is pBR322 and wherein the DNA sequence comprises:

(a) a first sequence ATG ACT GCT CTC TTT CTG ATG TCC ATG CTT TTT GGC CTT GCA TGT GGG CAA GCG ATG TCT TTT TGT ATT CCA ACT GAG TAT ACA ATG CAC ATC GAA AGG AGA GAG TGT GCT TAT TGC CTA ACC ATC AAC ACC ACC ATC TGT GCT GGA TAT TGT ATG ACA CGG;

(b) a second sequence of about 400-450 nucleotides following the first sequence; and

(c) a third sequence GAT ATC AAT GGC AAA CTG TTT CTT CCC AAA TAT GCT CTG TCC CAG GAT GTT TGC ACA TAT AGA GAC TTC ATC TAC AGG ACT GTA GAA ATA CCA GGA TGC CCA CTC CAT GTT GCT CCC TAT TTT TCC TAT CCT GTT GCT TTA AGC TGT AAG TGT GGC AAG TGC AAT ACT GAC TAT AGT GAC TGC ATA CAT GAA GCC ATC AAG ACA AAC TAC TGT ACC AAA CCT CAG AAG TCT TAT CTG GTA GGA TTT TCT GTC TAA following the second sequence.

5. The substantially pure human thyroid stimulating hormone of claim 4, wherein the plasmid is the pBR322 plasmid having ATCC Designation No. 67297.

6. The substantially pure human thyroid stimulating hormone of claim 4, wherein the plasmid is the pBR322 plasmid having ATCC Designation No. 67298.

7. Substantially pure human thyroid stimulating hormone comprising a beta subunit produced by a vector comprising a DNA sequence encoding said subunit, wherein the DNA sequence comprises:

(a) a first sequence ATG ACT GCT CTC TTT CTG ATG TCC ATG CTT TTT GGC CTT GCA TGT GGG CAA GCG ATG TCT TTT TGT ATT CCA ACT GAG TAT ACA ATG CAC ATC GAA AGG AGA GAG TGT GCT TAT TGC CTA ACC ATC AAC ACC ACC ATC TGT GCT GGA TAT TGT ATG ACA CGG;

(b) a second sequence of about 400-450 nucleotides following the first sequence; and

(c) a third sequence GAT ATC AAT GGC AAA CTG TTT CTT CCC AAA TAT GCT CTG TCC CAG GAT GTT TGC ACA TAT AGA GAC TTC ATC TAC AGG ACT GTA GAA ATA CCA GGA TGC CCA CTC CAT GTT GCT CCC TAT TTT TCC TAT CCT GTT GCT TTA AGC TGT AAG TGT GGC AAG TGC AAT ACT GAC TAT AGT GAC TGC ATA CAT GAA GCC ATC AAG ACA AAC TAC TGT ACC AAA CCT CAG AAG TCT TAT CTG GTA GGA TTT TCT GTC TAA following the second sequence.

8. The substantially pure human thyroid stimulating hormone of claim 4 or 7, wherein the beta subunit-encoding DNA sequence comprises the nucleotide sequence:

ATG ACT GCT CTC TTT CTG ATG TCC ATG CTT TTT GGC CTT GCA TGT GGG CAA GCG ATG TCT TTT TGT ATT CCA ACT GAG TAT ACA ATG CAC ATC GAA AGG AGA GAG TGT GCT TAT TGC CTA ACC ATC AAC ACC ACC ATC TGT GCT GGA TAT TGT ATG ACA CGG.

9. The substantially pure human thyroid stimulating hormone of claim 4 or 7, wherein the beta subunit-encoding DNA sequence comprises the nucleotide sequence:

GAT ATC AAT GGC AAA CTG TTT CTT CCC AAA TAT GCT CTG TCC CAG GAT GTT TGC ACA TAT AGA GAC TTC ATC TAC AGG ACT GTA GAA ATA CCA GGA TGC CCA CTC CAT GTT GCT CCC TAT TTT TCC TAT CCT GTT GCT TTA AGC TGT AAG TGT GGC AAG TGC AAT ACT GAC TAT AGT GAC TGC ATA CAT GAA GCC ATC AAG ACA AAC TAC TGT ACC AAA CCT CAG AAG TCT TAT CTG GTA GGA TTT TCT GTC TAA.
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