Nucleic acids involved in the responder phenotype and applications thereof

Abstract

The present invention relates to nucleic acid molecules encoding expression products involved in the Responder function, which contributes to the phenomenon of transmission ratio distortion. The present invention also relates to regulatory regions of the genes corresponding to said nucleic acid molecules. The present invention further relates to recombinant DNA molecules and vectors comprising said nucleic acid molecules and/or regulatory regions as well as to host cells transformed therewith. Additionally, the present invention relates to transgenic animals, comprising said nucleic acid molecules, recombinant DNA molecules or vectors stably integrated into their genome. The various embodiments of the invention have a significant impact on breeding strategies by allowing for the specific selection of genetic traits and in particular of sex. Further, the present invention finds applications in the development of contraceptiva.

1 . A nucleic acid molecule comprising a transcription unit encoding in its 5′-portion a kinase having a homology to the MARK2 kinase whereas the 3′-portion of the nucleotide sequence has a high homology to the rsk3 kinase.  
     
     
         2 . A nucleic acid molecule, preferably according to  claim 1 , encoding an expression product involved in the Responder phenotype, which contributes to the phenomenon of transmission ratio distortion, selected from the group consisting of 
 (a) a nucleic acid molecule comprising the nucleic acid molecule as shown in FIGS. 4 a,    9 ,  7   a,    7   b,    7   c,    7   d  or a fragment thereof;    (b) a nucleic acid molecule being an allelic variant or a homologue of the nucleic acid molecule of (a);    (c) a nucleic acid molecule hybridizing to a nucleic acid molecule complementary to the nucleic acid molecule of (a) or (b); and    (d) a nucleic acid molecule which is related to the nucleic acid molecule of (a), (b) or (c) by the degeneration of the genetic code.    
     
     
         3 . The nucleic acid molecule of  claim 1  or  2  which is a DNA molecule.  
     
     
         4 . The nucleic acid molecule of any one of  claims 1  to  3 , wherein said expression product is an RNA or a (poly)peptide.  
     
     
         5 . The nucleic acid molecule of any one of  claims 1  to  4 , wherein said Responder function is the mouse-t-complex Responder function.  
     
     
         6 . A regulatory region of the gene corresponding to the nucleic acid molecule defined in any one of  claims 1  to  5  being capable of controlling expression of said nucleic acid molecule.  
     
     
         7 . The regulatory region of  claim 6  which is a naturally occurring regulatory region or a genetically engineered derivative thereof.  
     
     
         8 . The regulatory region of  claim 6  or  7  which comprises or is a promoter.  
     
     
         9 . The regulatory region of  claim 8  which comprises the fragment from nucleotides 930 to 3576 of the sequence shown in FIG. 11.  
     
     
         10 . A recombinant DNA molecule comprising the nucleic acid molecule of any one of  claims 1  to  5  and/or the regulatory region of any one of  claims 6  to  9 , and/or a regulatory region allowing expression during spermatogenesis/spermiogenesis.  
     
     
         11 . The recombinant DNA molecule of  claim 10 , wherein said regulatory region is operatively linked to a heterologous DNA molecule.  
     
     
         12 . The recombinant DNA molecule of  claim 11 , wherein said heterologous DNA molecule encodes a peptide, a polypeptide, an antisense RNA, a sense RNA, a toxin and/or a ribozyme.  
     
     
         13 . The recombinant DNA molecule of  claim 12  wherein said peptide, polypeptide, antisense RNA, sense RNA, toxin and/or ribozyme is capable of causing cell death.  
     
     
         14 . The recombinant DNA molecule of  claim 12  wherein said or an additional peptide or polypeptide is an effector (poly)peptide.  
     
     
         15 . The recombinant DNA of  claim 14 , wherein said effector (poly)peptide is capable of sequestering an ion selectively binding to a solid support, or binding to a preselected antigenic determinant or is a toxin, a ribozyme, an enzyme, a label or a remotely detectable moiety.  
     
     
         16 . The recombinant DNA of  claim 15 , wherein said effector (poly)peptide is calmodulin, methallothionein, a fragment thereof, green fluorescent protein (GFP), β-lactamase, hCD24, myc, FLAG, hemagglutinin or an amino acid sequence rich in at least one of glutamic acid, aspartic acid, lysine, histidine or arginine.  
     
     
         17 . A vector comprising the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9  or the recombinant DNA molecule of any one of  claims 10  to  16 .  
     
     
         18 . The vector of  claim 17 , comprising a heterologous promoter.  
     
     
         19 . The vector of  claim 18 , wherein the heterologous promoter is controlling gene expression in spermatogenesis and/or in spermiogenesis.  
     
     
         20 . The vector of  claim 19 , wherein the heterologous promoter is the testis promoter of c-kit or of ACE.  
     
     
         21 . A host cell or organism transformed or transfected with the nucleic acid molecule of any one of  claims 1  to  5 , the recombinant DNA molecule of any one of  claims 10  to  16  or the vector of any one of  claims 17  to  20 .  
     
     
         22 . A method of recombinantly producing an expression product as defined in any one of  claims 1  to  20  comprising the steps of culturing the host cell of  claim 21  under conditions to cause expression of the protein and recovering said protein from the culture.  
     
     
         23 . An expression product encoded by the nucleic acid molecule of any one of  claims 1  to  20  or obtainable by the method of  claim 22 .  
     
     
         24 . An antibody specifically recognizing the expression product of  claim 23 .  
     
     
         25 . A nucleic acid molecule specifically hybridizing with the nucleic acid molecule of any one of  claims 1  to  5  translatable into said MARK related kinase or to an intron of said nucleic acid molecule or with the regulatory region of any one of  claims 6  to  9  or with a complementing strand thereof.  
     
     
         26 . A pharmaceutical composition comprising the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20 , the host cell of  claim 21 , the expression product of  claim 23 , the antibody of  claim 24  or the nucleic acid molecule of  claim 25 .  
     
     
         27 . A diagnostic composition comprising the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20 , the host cell of  claim 21 , the expression product of  claim 23 , the antibody of  claim 24  or the nucleic acid molecule of  claim 25  or a pair of primers wherein one of said primers is the nucleic acid molecule of  claim 25 .  
     
     
         28 . A method for the production of a transgenic non human mammal, fish or bird comprising introducing the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA molecule of any one of  claims 10  to  16  or the vector of any one of  claims 17  to  20  into the chromosome of a germ cell, embryonic cell or an egg cell or a cell derived therefrom.  
     
     
         29 . The method of  claim 28  wherein said chromosome is an X chromosome or the corresponding sex chromosome in birds or fish or an autosome.  
     
     
         30 . The method of  claim 28  wherein said chromosome is a Y chromosome or the corresponding sex chromosome in birds or fish.  
     
     
         31 . The method of  claim 30  wherein the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA molecule of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20 , a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined in  claim 14  or  15  is/are integrated in said Y chromosome in a reversible inactive state of expressibility.  
     
     
         32 . The method of  claim 31  wherein said nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA molecule of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20 , a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined in  claim 14  or  15  alone or in combination is/are flanked by lox P sites or FRT sites.  
     
     
         33 . The method of any one of  claims 28  to  32  further comprising introducing a nucleic acid molecule encoding at least one Distorter into the same or a different chromosome or introducing a chromosomal fragment comprising at least one Distorter into said cell.  
     
     
         34 . The method of  claim 33  wherein said Distorter is D2 and/or D1.  
     
     
         35 . A method for the production of a male transgenic non human mammal, fish or bird having integrated in its Y chromosome the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA molecule of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20 , a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined in any one of  claims 14  to  16  in an active state of expressibility, said method comprising in vitro fertilisation using sperm from said male transgenic non human mammal, fish or bird.  
     
     
         36 . The method of  claim 35 , prior to in vitro fertilisation further comprising allowing expression of said effector (poly)peptide and selecting for sperm expressing said effector (poly)peptide and, thus, containing said Y chromosome.  
     
     
         37 . A transgenic non human mammal, fish or bird having stably integrated into its genome the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA molecule of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20  or which regenerated from a host cell of  claim 21 , or which is obtainable by the method of any one of  claims 28  to  36 .  
     
     
         38 . A pair of transgenic non human mammals, fish or bird, wherein the male is a transgenic animal as defined in any one of  claims 31  to  36 , and the female is a transgenic animal having stably integrated into its genomic DNA a nucleic acid molecule encoding a site specific DNA recombinase.  
     
     
         39 . The pair of animals of  claim 38 , wherein said DNA recombinase is Cre or flp.  
     
     
         40 . The pair of animals of  claim 38  or  39 , wherein said DNA recombinase is controlled by regulatory elements that are active prior to spermiogenesis.  
     
     
         41 . Sperm obtainable from a male of the transgenic non-human mammal, fish or bird as defined in any one of  claims 37  to  40 .  
     
     
         42 . A method for the selection of sperm of  claim 41  comprising allowing expression of the effector (poly)peptide and selecting for the presence or absence of said (poly)peptide.  
     
     
         43 . A method for the selection against sperm of  claim 42  comprising 
 (a) allowing expression of the recombinant DNA molecule of  claim 13;  and  
 (b) selecting for viable sperm.  
 
     
     
         44 . Use of the sperm of  claim 41  or of sperm obtainable by the method of  claim 42  or  43  for the production of offspring.  
     
     
         45 . Use of the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA of any one of claims  10  to  16 , the vector of any one of  claims 17  to  20 , the host cell of  claim 21  the expression product of  claim 23 , the antibody of  claim 24  or the nucleic acid molecule of  claim 25  for the isolation of receptors on the surface of sperm recognizing attractants of the egg cell for the development and/or production of contraceptiva.  
     
     
         46 . Use of the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20 , the host cell of  claim 21 , the expression product of  claim 23 , the antibody of  claim 24  or the nucleic acid molecule of  claim 25  for the identification of chemicals or biological compounds able to trigger the (premature) activation or inhibition of the Responder/Distorter signaling cascade.  
     
     
         47 . Use of the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20 , the host cell of  claim 21 , the expression product of  claim 23 , the antibody of  claim 24  or the nucleic acid molecule of  claim 25  for the isolation of receptor molecules and/or other members of the Responder/Distorter signaling cascade to which said expression product may bind.  
     
     
         48 . A method for the detection of the nucleic acid molecule of any one of  claims 1  to  5 , the regulatory region of any one of  claims 6  to  9 , the recombinant DNA molecule of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20  the transgenic non human mammal, fish or bird of  claim 37  or a part thereof comprising identifying the nucleic acid molecule of any one of  claims 1  to  5 , the recombinant DNA molecule of any one of  claims 10  to  16 , the vector of any one of  claims 17  to  20  or a portion thereof, or the expression product of the invention or a different expression product encoded by said DNA molecule or vector in said transgenic animal or a part thereof.  
     
     
         49 . A method of distorting the transmission ratio of genetic traits comprising manipulating the sequence or expression level of a different member of the Responder/Distorter signal cascade than the t-Responder, and restricting the expression of the manipulated form of said different member preferentially or completely to those sperm carrying it.  
     
     
         50 . A transgenic animal having an recombinantly manipulated altered sequence or expression level of a member of the Responder/Distorter signal cascade, and wherein the expression of said member has been restricted preferentially or completely to those sperm carrying it.  
     
     
         51 . The transgenic animal of  claim 50  wherein said member is not the Responder.  
     
     
         52 . A method for the distortion, to a non-Mendelian ratio, of the transmission of a genetic trait from male mammals to their offspring comprising expressing during spermatogenesis/spermiogenesis a gene involved in sperm motility and/or fertilization.  
     
     
         53 . The method of  claim 52 , wherein said genetic trait determines the sex.  
     
     
         54 . The method of  claim 52  or  53 , wherein said gene is under the control of a promoter that allows expression during spermatogenesis/spermiogenesis.  
     
     
         55 . The method of  claim 54 , wherein said promoter allows the preferential or exclusive expression of said gene in sperm carrying said gene.  
     
     
         56 . The method of any one of  claims 52  to  55 , wherein said gene is engineered such as to interfere with the function of its wild type allele or with the function of other genes involved in sperm motility and/or fertilization, wherein said gene inhibits the function of one or more genes involved in sperm motility and/or fertilization, and/or wherein said gene causes cell death in spermatocytes/spermatids expressing it, and/or wherein said gene encodes a tag allowing the in vitro selection of sperm carrying said tag.  
     
     
         57 . The method of any one of  claims 52  to  56 , wherein said gene encodes an inhibitor of cAMP dependent protein kinase A.  
     
     
         58 . The method of  claim 57 , wherein said inhibitor is PKI or a functionally active derivative or fragment thereof.  
     
     
         59 . A transgenic animal comprising a gene as defined in any one of  claims 52  to  58 .  
     
     
         60 . Sperm obtainable from the transgenic animal of  claim 59 .  
     
     
         61 . An isolated nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent hybridization conditions to a complement of the nucleotide sequence as shown in FIG. 4 a,  wherein the nucleic acid molecule encodes an expression product having kinase activity.  
     
     
         62 . A vector comprising the nucleic acid molecule of  claim 61 .  
     
     
         63 . A recombinant DNA molecule comprising the nucleic acid molecule of  claim 61 .  
     
     
         64 . A vector comprising the recombinant DNA molecule of  claim 63 .  
     
     
         65 . The vector of  claim 62  or  64  wherein the vector is an expression vector.  
     
     
         66 . A host cell comprising the isolated nucleic acid molecule of  claim 61 .  
     
     
         67 . A pharmaceutical composition comprising the isolated nucleic acid molecule of  claim 61 .  
     
     
         68 . A method comprising culturing the host cell of  claim 66  under conditions to cause expression of an expression product encoded by the isolated nucleic acid.  
     
     
         69 . An expression product encoded by the isolated nucleic acid molecule of  claim 61 .  
     
     
         70 . A host cell comprising the expression product of  claim 69 .  
     
     
         71 . A pharmaceutical composition comprising the expression product of  claim 69.