Survivin, a protein that inhibits cellular apoptosis, and its modulation

Abstract

The present invention provides the amino acid of a protein that inhibits cellular apoptosis, herein termed the Survivin protein and nucleic acid molecules that encode Survivin. Based on this disclosure, the present invention provides isolated Survivin protein, isolated Survivin encoding nucleic acid molecules, methods of isolating other members of the Survivin family of proteins, methods for identifying agents that block Survivin mediated inhibition of cellular apoptosis, methods of using agents that block Survivin mediated inhibition or Survivin expression to modulate biological and pathological processes, and methods of assaying Survivin activity.

1 - 59 . (canceled)  
     
     
         60 . An oligonucleotide that hybridizes under stringent conditions with a polynucleotide having the sequence SEQ ID NO: 35, but does not hybridize under stringent conditions with a polynucleotide having the sequence of human EPR-1 mRNA, wherein the stringent conditions are selected from the group consisting of: (1) washing with 0.015 M sodium chloride, 0.0015 M sodium titrate, 0.1% SDS at 50° C.; (2) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; and (3) hybridization in 50% formamide, 5×SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS, or the complement of said oligonucleotide.  
     
     
         61 . The oligonucleotide of  claim 60  or its complement, wherein the oligonucleotide is a DNA oligonucleotide.  
     
     
         62 . The oligonucleotide of  claim 60  or its complement, wherein the oligonucleotide is extendable by a polymerase enzyme.  
     
     
         63 . The oligonucleotide of  claim 60  or its complement, comprising a detectable label.  
     
     
         64 . A kit for amplifying an mRNA encoding survivin, the kit comprising a first extendable primer that hybridizes under stringent conditions with the mRNA and at least one second primer that hybridizes under stringent conditions with an antisense strand of the mRNA, wherein at least one of the first and second primers is an oligonucleotide of  claim 60  or its complement.  
     
     
         65 . A kit for amplifying an mRNA encoding survivin, the kit comprising a first extendable primer that hybridizes under stringent conditions with the mRNA and at least one second primer that hybridizes under stringent conditions with an antisense strand of the mRNA, wherein each of the first and second primers is an oligonucleotide of  claim 60  or a complement thereof.  
     
     
         66 . A method of detecting expression of survivin in a sample obtained from a human, the method comprising using the oligonucleotide of  claim 60 , or its complement, as a primer in a reverse-transcriptase amplification method for amplifying mRNA encoding survivin.  
     
     
         67 . An oligonucleotide that hybridizes under stringent conditions with a polynucleotide having the sequence of the coding sequence of survivin, but does not hybridize under stringent conditions with a polynucleotide having the sequence of the coding sequence of human EPR-1, wherein the stringent conditions are selected from the group consisting of: (1) washing with 0.015 M sodium chloride, 0.0015 M sodium titrate, 0.1% SDS at 50° C.; (2) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; and (3) hybridization in 50% formamide, 5×SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS, or the complement of said oligonucleotide.  
     
     
         68 . The oligonucleotide of  claim 67  or its complement, wherein the oligonucleotide is a DNA oligonucleotide.  
     
     
         69 . The oligonucleotide of  claim 67  or its complement, wherein the oligonucleotide is extendable by a polymerase enzyme.  
     
     
         70 . The oligonucleotide of  claim 67  or its complement, comprising a detectable label.  
     
     
         71 . The oligonucleotide of  claim 67  or its complement, wherein the coding sequence of survivin has the sequence of nucleotides 2811-2921 directly attached to nucleotides 3174-3283 directly attached to nucleotides 5158-5275 directly attached to nucleotides 11955-12044 of SEQ ID NO: 35.  
     
     
         72 . A kit for amplifying an mRNA encoding survivin, the kit comprising a first extendable primer that hybridizes under stringent conditions with the mRNA and at least one second primer that hybridizes under stringent conditions with an antisense strand of the mRNA, wherein at least one of the first and second primers is an oligonucleotide of  claim 67  or its complement.  
     
     
         73 . A kit for amplifying an mRNA encoding survivin, the kit comprising a first extendable primer that hybridizes under stringent conditions with the mRNA and at least one second primer that hybridizes under stringent conditions with an antisense strand of the mRNA, wherein each of the first and second primers is an oligonucleotide of  claim 67  or a complement thereof.  
     
     
         74 . A method of detecting expression of survivin in a sample obtained from a human, the method comprising using the oligonucleotide of  claim 67 , or its complement, as a primer in a reverse-transcriptase amplification method for amplifying mRNA encoding survivin.  
     
     
         75 . An oligonucleotide that hybridizes under stringent conditions with the mRNA encoding human survivin, but does not hybridize under stringent conditions with the mRNA encoding human EPR-1, wherein the stringent conditions are selected from the group consisting of: (1) washing with 0.015 M sodium chloride, 0.0015 M sodium titrate, 0.1% SDS at 50° C.; (2) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; and (3) hybridization in 50% formamide, 5×SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS, or the complement of said oligonucleotide.  
     
     
         76 . The oligonucleotide of  claim 75  or its complement, wherein the oligonucleotide is a DNA oligonucleotide.  
     
     
         77 . The oligonucleotide of  claim 75  or its complement, wherein the oligonucleotide is extendable by a polymerase enzyme.  
     
     
         78 . The oligonucleotide of  claim 75  or its complement, comprising a detectable label.  
     
     
         79 . A kit for amplifying an mRNA encoding survivin, the kit comprising a first extendable primer that hybridizes under stringent conditions with the mRNA and at least one second primer that hybridizes under stringent conditions with an antisense strand of the mRNA, wherein at least one of the first and second primers is an oligonucleotide of  claim 75  or its complement.  
     
     
         80 . A kit for amplifying an mRNA encoding survivin, the kit comprising a first extendable primer that hybridizes under stringent conditions with the mRNA and at least one second primer that hybridizes under stringent conditions with an antisense strand of the mRNA, wherein each of the first and second primers is an oligonucleotide of  claim 75  or a complement thereof.  
     
     
         81 . A method of detecting expression of survivin in a sample obtained from a human, the method comprising using the oligonucleotide of  claim 75 , or its complement, as a primer in a reverse-transcriptase amplification method for amplifying mRNA encoding survivin.