Purified and isolated piwi family genes and gene products and methods using same

Abstract

A family of isolated and purified proteins and nucleic acids are disclosed. Particularly, piwi family proteins and cDNAs encoding the same are disclosed. Recombinant host cells, recombinant nucleic acids, recombinant proteins and transgenic animals are also disclosed, along with methods of producing each. Isolated and purified antibodies to piwi family homologs, and methods of producing the same, are also disclosed. piwi family gene products are characterized as having activity in the growth, proliferation and self-renewing division of stem cells, and proliferation of primordial germ cells. Thus, therapeutic, screening, culturing and transgenic methods involving these activities are also disclosed.

1 . An isolated and purified biologically active piwi family polypeptide comprising: 
 (a) a polypeptide encoded by a nucleic acid sequence as set forth in any of SEQ ID NOs:1, 3 and 5;    (b) a polypeptide encoded by a nucleic acid having at least about 75% or greater homology to a DNA sequence as set forth in any of SEQ ID NOs:1, 3 and 5;    (c) a polypeptide encoded by a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs:1, 3 and 5;    (d) a polypeptide which is a biologically functional equivalent of a peptide as set forth in any of SEQ ID NOs:2, 4 and 6;    (e) a polypeptide which is immunologically cross-reactive with antibodies which are immunologically reactive with peptides encoded by the nucleic acid sequences as set forth in any of SEQ ID NOs:2, 4 and 6;    (f) a polypeptide encoded by a nucleic acid molecule capable of hybridizing to a nucleic acid molecule having the sequence of any of SEQ ID NOs:1, 3 and 5, or fragments or variants or complementary sequences thereof, under high stringency conditions; or    (g) a polypeptide comprising a portion of a polypeptide of a), b), c), d), e), or f).    
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide comprises a vertebrate piwi family polypeptide.  
     
     
         3 . The polypeptide of  claim 2 , wherein the polypeptide comprises a human piwi family polypeptide.  
     
     
         4 . The polypeptide of  claim 1 , wherein the piwi family comprises an amino acid sequence as set forth in any of SEQ ID NOs:2, 4 and 6.  
     
     
         5 . The polypeptide of  claim 1 , modified to be in detectably labeled form.  
     
     
         6 . An isolated and purified antibody capable of specifically binding to the polypeptide of  claim 1 .  
     
     
         7 . The antibody of  claim 6  which is a monoclonal antibody.  
     
     
         8 . The antibody of  claim 6  which is a polyclonal antibody.  
     
     
         9 . A hybridoma cell line which produces the monoclonal antibody of  claim 8 .  
     
     
         10 . An isolated and purified antibody capable of neutralizing the biological activity of the polypeptide of  claim 1 .  
     
     
         11 . The antibody of  claim 10  which is a monoclonal antibody.  
     
     
         12 . The antibody of  claim 10  which is a polyclonal antibody.  
     
     
         13 . A hybridoma cell line which produces the monoclonal antibody of  claim 11 .  
     
     
         14 . An isolated and purified polynucleic acid encoding a biologically active piwi family polypeptide of  claim 1 .  
     
     
         15 - 29 . (Canceled).  
     
     
         30 . A method of detecting in a sample an RNA that encodes the piwi family polypeptide encoded by the nucleic acid of  claim 14 , said method comprising the steps of: 
 (a) contacting said sample under hybridizing conditions with the polynucleic acid of  claim 14  to form a duplex; and    (b) detecting the presence of said duplex.    
     
     
         31 . A method of producing an antibody immunoreactive with a piwi family polypeptide, the method comprising steps of: 
 (a) transfecting a recombinant host cell with the a polynucleic acid of  claim 14 , which encodes a piwi family polypeptide;    (b) culturing the host cell under conditions sufficient for expression of the polypeptide;    (c) recovering the polypeptide; and    (d) preparing the antibody to the polypeptide.    
     
     
         32 . The method of  claim 31 , wherein the polypeptide comprises a polypeptide as essentially set forth in any of SEQ ID NOs:2, 4 and 6.  
     
     
         33 . The method of  claim 31 , wherein the polynucleic acid comprises a polynucleic acid sequence as essentially set forth in any of SEQ ID NOs:1, 3 and 5.  
     
     
         34 . An antibody produced by the method of  claim 31 .  
     
     
         35 . A method of detecting a piwi family polypeptide, the method comprising immunoreacting the polypeptide with an antibody prepared according the method of  claim 31  to form an antibody-polypeptide conjugate; and detecting the conjugate.  
     
     
         36 . A method of detecting a messenger RNA transcript that encodes a piwi family polypeptide, the method comprising the steps of hybridizing the messenger RNA transcript with the polynucleic acid of  claim 14  to form a duplex; and detecting the duplex.  
     
     
         37 . A method of detecting a DNA molecule that encodes a piwi family polypeptide, the method comprising the steps of hybridizing DNA molecules with the polynucleic acid of  claim 14  to form a duplex; and detecting the duplex.  
     
     
         38 . An assay kit for detecting the presence of a piwi family polypeptide in a biological sample, the kit comprising a first container containing a first antibody capable of immunoreacting with a piwi family polypeptide of  claim 1 , wherein the first antibody is present in an amount sufficient to perform at least one assay.  
     
     
         39 . The assay kit of  claim 38 , further comprising a second container containing a second antibody that immunoreacts with the first antibody.  
     
     
         40 . The assay kit of  claim 39 , wherein the first antibody and the second antibody comprise monoclonal antibodies.  
     
     
         41 . The assay kit of  claim 39 , wherein the first antibody is affixed to a solid support.  
     
     
         42 . The assay kit of  claim 39 , wherein the first and second antibodies each comprise an indicator.  
     
     
         43 . The assay kit of  claim 42 , wherein the indicator is a radioactive label, a fluorescent label or an enzyme.  
     
     
         44 . An assay kit for detecting the presence, in biological samples, of a piwi family polypeptide, the kit comprising a first container that contains a polynucleic acid identical or complimentary to a segment of at least ten contiguous nucleotide bases of the polynucleic acid of  claim 14 .  
     
     
         45 . An assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a piwi family polypeptide, the kit comprising a first container containing a piwi family polypeptide of  claim 1  that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.  
     
     
         46 . A method of screening candidate substances for an ability to modulate piwi family biological activity, the method comprising the steps of: 
 (a) establishing replicate test and control samples that comprise a biologically active piwi family polypeptide;    (b) administering a candidate substance to the test sample but not the control sample;    (c) measuring piwi family biological activity in the test and the control samples; and    (d) determining that the candidate substance modulates piwi family biological activity if the piwi family biological activity measured for the test sample is greater or less than the piwi family biological activity measured for the control sample.    
     
     
         47 . The method of  claim 46 , wherein the candidate substance is further characterized as a candidate polypeptide, and further comprising the step of purifying and isolating a gene encoding the candidate polypeptide.  
     
     
         48 . The method of  claim 46 , wherein the replicate test and control samples further comprise a cell that expresses a biologically active vertebrate piwi family polypeptide.  
     
     
         49 . (Canceled).  
     
     
         50 . A method of modulating piwi family polypeptide activity in a subject, the method comprising the step of administering to the subject an effective amount of a substance capable of modulating the piwi family polypeptide activity in the subject, whereby modulation of the piwi family polypeptide activity is accomplished.  
     
     
         51 . The method of  claim 50 , wherein the step of administering further comprises administering an effective amount of a substance that modulates expression of a piwi family-encoding polynucleic acid in the subject.  
     
     
         52 . The method of  claim 51 , wherein the substance that modulates expression of a piwi family-encoding polynucleic acid comprises an antisense oligonucleotide.  
     
     
         53 . The method of  claim 50 , where the substance that modulates the piwi family activity comprises an anti-piwi family antibody.  
     
     
         54 . The method of  claim 53 , where the anti-piwi family antibody comprises a monoclonal antibody.  
     
     
         55 . The method of  claim 50 , wherein the piwi family activity comprises modulating cell growth, proliferation, division and combinations thereof, and wherein the step of administering comprises administering to the subject an effective piwi family-modulating amount of a piwi family-modulating substance.  
     
     
         56 . The method of  claim 50 , wherein the subject is a vertebrate.  
     
     
         57 . The method of  claim 56 , wherein the vertebrate is a warm-blooded vertebrate.  
     
     
         58 . A method of treating a subject suffering from a disorder associated with piwi family biological activity, the method comprising: administering to the subject an effective amount of a piwi family biological activity-modulating substance, whereby treatment of the disorder is accomplished.  
     
     
         59 . The method of  claim 58 , wherein the step of administering further comprises administering an effective amount of a substance that modulates expression of a piwi family-encoding polynucleic acid in the subject.  
     
     
         60 . The method of  claim 59 , wherein the substance that modulates expression of a piwi family-encoding polynucleic acid comprises an antisense oligonucleotide.  
     
     
         61 . The method of  claim 58 , wherein the substance capable of modulating piwi family activity in the subject comprises an anti-piwi family antibody.  
     
     
         62 . The method of  claim 61 , wherein the anti-piwi family antibody comprises a monoclonal antibody.  
     
     
         63 . A method of treating a subject suffering from a disorder associated with piwi-mediated biological activity, the method comprising administering to the subject a therapeutic composition which comprises a biologically active piwi family polypeptide, whereby treatment of disorder associated with piwi-mediated biological activity in the subject is accomplished.  
     
     
         64 . The method of  claim 63 , wherein the therapeutic composition comprises a biologically active piwi family polypeptide and a pharmaceutically acceptable carrier.  
     
     
         65 . The method of  claim 63 , wherein the piwi-mediated biological activity comprises modulation of stem cell growth, proliferation, division and combinations thereof, modulation of primordial germ cell proliferation, and combinations thereof; and the piwi family polypeptide is administered in an effective amount.  
     
     
         66 . A method for modulating piwi-mediated biological activity in a cell, the method comprising: 
 (a) delivering to the cell an effective amount of a DNA molecule comprising a polynucleotide that encodes a biologically active piwi family polypeptide; and    (b) maintaining the cell under conditions sufficient for expression of said polypeptide.    
     
     
         67 . The method of  claim 66 , wherein the polypeptide comprises a polypeptide as essentially set forth in any of SEQ ID NOs:2, 4 and 6.  
     
     
         68 . The method of  claim 67 , wherein the polypeptide is encoded by a polynucleic acid as essentially set forth in any of SEQ ID NOs:1, 3 and 5.  
     
     
         69 . A method of culturing a primitive cell, the method comprising: 
 (a) providing a culture comprising a primitive cell; and    (b) delivering to the primitive cell a piwi family gene product, whereby growth of the primitive cell in a culture is accomplished.    
     
     
         70 . The method of  claim 69 , wherein the primitive cell is maintained in the culture for a time sufficient to produce a sustained cell culture.  
     
     
         71 . The method of  claim 69 , wherein the piwi family gene product is delivered to the primitive cell by delivering to the primitive cell an effective amount of a polynucleotide that encodes a biologically active piwi family polypeptide; and maintaining the primitive cell under conditions sufficient for expression of the polypeptide.  
     
     
         72 . The method of  claim 69 , wherein the piwi family gene product is delivered to the cell by administration of a biologically active piwi family polypeptide to the culture.  
     
     
         73 . The method of  claim 69 , further comprising: 
 (a) collecting primitive cells;    (b) depositing the primitive cells in contact with a feeder matrix, the feeder matrix comprising a piwi family gene product; and    (c) culturing the primitive cells on the feeder matrix in the presence of media for a time sufficient to produce a sustained primitive cell culture.    
     
     
         74 . The method of  claim 69 , wherein the feeder matrix comprises a recombinant cell comprising a polynucleic acid encoding a biologically active piwi family polypeptide.  
     
     
         75 . The method of  claim 69 , further comprising establishing a monolayer of cells in the feeder matrix.  
     
     
         76 . A transgenic or chimeric non-human animal having incorporated into its genome a polynucleic acid encoding a biologically active piwi family polypeptide, the polynucleic acid being present in said genome in a copy number effective to confer expression in the animal of the piwi family polypeptide.  
     
     
         77 . The transgenic or chimeric non-human animal of  claim 76 , wherein said polynucleic acid comprises a human piwi family-encoding segment.  
     
     
         78 . The transgenic or chimeric non-human animal of  claim 76 , wherein the expression of the piwi family polypeptide is conferred in reproductive tissue of the animal.  
     
     
         79 . The transgenic or chimeric non-human animal of  claim 76 , wherein the expression of the piwi family polypeptide confers an increased number of germline stem cells or primordial germ cells in the animal as compared to a normal animal.  
     
     
         80 . The transgenic or chimeric non-human animal of  claim 76 , further comprising an additional polynucleic acid encoding a predetermined biologically active polypeptide, the polynucleic acid being present in said genome in a copy number effective to confer expression in the animal of the polypeptide.  
     
     
         81 . A method of altering the phenotype of an embryonic animal, the method comprising: 
 (a) providing a recombinant primitive cell comprising a polynucleic acid encoding a biologically active piwi family polypeptide;    (b) transfecting the recombinant primitive cell with a polynucleic acid encoding a predetermined biologically active polypeptide;    (c) transferring the transfected primitive cells into an embryo to confer expression of the predetermined biologically active polypeptide animal of the polypeptide, whereby the phenotype of the embryo is altered.    
     
     
         82 . The method of  claim 81 , wherein the animal is a bird, and the transfected primitive cell is transferred into an egg containing an embryonic bird.  
     
     
         83 . The method of  claim 81 , wherein the altered phenotype comprises a change in protein expression, a change in growth rate, feed efficiency, a change in stem cell count, a change in primordial germ cell count, disease resistance, or a combination thereof.  
     
     
         84 . The method of  claim 81 , wherein the embryonic animal is permitted to develop into a fetus or animal.  
     
     
         85 . A method of recovering a protein from a transgenic or chimeric non-human animal, the method comprising: 
 (a) providing a transgenic or chimeric non-human animal having incorporated into its genome a polynucleic acid encoding a biologically active piwi family polypeptide, the polynucleic acid being present in said genome in a copy number effective to confer expression in the animal of the piwi family polypeptide; and    (b) recovering the biologically active piwi family polypeptide from the animal provided in step (a).    
     
     
         86 . The method of  claim 85 , wherein the transgenic or chimeric non-human animal provided in step (a) further comprises another polynucleic acid incorporated into its genome, the polynucleic acid encoding a predetermined biologically active polypeptide, the polynucleic acid being present in said genome in a copy number effective to confer expression in the animal of the predetermined polypeptide; and wherein the predetermined biologically active polypeptide is recovered in addition to or instead of the biologically active piwi family polypeptide.  
     
     
         87 . The method of  claim 85  or  86 , wherein the transgenic or chimeric non-human animal provided in step (a) further comprises an avian, and the polypeptide is recovered from an egg produced by the avian.  
     
     
         88 . A pharmaceutical composition comprising an isolated and purified biologically active piwi family polypeptide and a pharmaceutically acceptable carrier.  
     
     
         89 . The pharmaceutical composition of  claim 88 , wherein the polypeptide comprises a polypeptide as essentially set forth in any of SEQ ID NOs:2, 4 and 6.  
     
     
         90 . The pharmaceutical composition of  claim 88 , wherein the polypeptide is encoded by a polynucleic acid sequence as essentially set forth in any of SEQ ID NOs:1, 3 and 5.  
     
     
         91 . The pharmaceutical composition of  claim 88 , further comprising a cell expressing the polypeptide.  
     
     
         92 . A pharmaceutical composition comprising an isolated and purified polynucleic acid encoding a biologically active piwi family polypeptide and a pharmaceutically acceptable carrier.  
     
     
         93 . The pharmaceutical composition of  claim 92 , wherein the encoded polypeptide comprises an amino acid sequence as essentially set forth in any of SEQ ID NOs:2, 4 and 6.  
     
     
         94 . The pharmaceutical composition of  claim 92 , further comprising a piwi family-encoding polynucleic acid sequence as essentially set forth in any of SEQ ID NOs:1, 3 and 5.  
     
     
         95 . The pharmaceutical composition of  claim 92 , wherein the polynucleic acid is a DNA segment.  
     
     
         96 . The pharmaceutical composition of  claim 92 , wherein the polynucleic acid is positioned under the control of a promoter.  
     
     
         97 . The pharmaceutical composition of  claim 92 , wherein the polynucleic acid further comprises a recombinant vector.  
     
     
         98 . The pharmaceutical composition of  claim 92 , further comprising a cell expressing the encoded polypeptide.