U.S. patent application number 10/454224 was filed with the patent office on 2004-01-15 for nucleic acids involved in the responder phenotype and applications thereof.
This patent application is currently assigned to Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.. Invention is credited to Herrmann, Bernhard, Kispert, Andreas, Koschorz, Birgit.
Application Number | 20040010814 10/454224 |
Document ID | / |
Family ID | 26145903 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040010814 |
Kind Code |
A1 |
Herrmann, Bernhard ; et
al. |
January 15, 2004 |
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.
Inventors: |
Herrmann, Bernhard;
(Freiburg, DE) ; Koschorz, Birgit; (Freiburg,
DE) ; Kispert, Andreas; (Freiburg, DE) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55458
US
|
Assignee: |
Max-Planck-Gesellschaft zur
Forderung der Wissenschaften e.V.
Berlin
DE
|
Family ID: |
26145903 |
Appl. No.: |
10/454224 |
Filed: |
June 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10454224 |
Jun 4, 2003 |
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09554726 |
Jul 26, 2000 |
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6642369 |
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09554726 |
Jul 26, 2000 |
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PCT/EP98/07395 |
Nov 18, 1998 |
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Current U.S.
Class: |
800/14 ; 435/194;
435/320.1; 435/353; 435/6.11; 435/6.16; 435/69.1; 536/23.2;
800/18 |
Current CPC
Class: |
A61K 38/00 20130101;
A01K 2217/05 20130101; A61K 39/00 20130101; A61P 15/16 20180101;
C12N 9/12 20130101; A61K 48/00 20130101 |
Class at
Publication: |
800/14 ; 435/6;
435/69.1; 435/353; 435/320.1; 536/23.2; 435/194; 800/18 |
International
Class: |
A01K 067/027; C12Q
001/68; C07H 021/04; C12N 009/12; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 18, 1997 |
EP |
97 12 0190.0 |
Mar 2, 1998 |
EP |
98 10 3596.7 |
Claims
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. 4a, 9, 7a, 7b, 7c, 7d 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), .beta.-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/spermiogenesi- s.
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. 4a,
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.
Description
[0001] 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.
[0002] The mouse T/t-complex, a region of approximately 12 cM
genetic distance on the proximal part of chromosome 17, contains
several loci acting in concert to produce a phenomenon called
transmission ratio distortion (TRD). The latter designation
indicates the fact that the so-called t-haplotype form of this
chromosomal region has a selective advantage over the wild type
form in that it is transmitted to the offspring at non-mendelian
ratios of up to 99%. This transmission at non-mendelian ratio is
achieved by the concerted action of mainly four loci, the so-called
Distorters Tcd-1 (D1), Tcd-2 (D2) and Tcd-3 (D3), and the Responder
Tcr (R.sup.t)(Lyon 1984). Two more Distorters have been postulated
by other authors (Silver and Remis 1987).
[0003] According to Lyon's model (Lyon 1986) which formally
explains the genetic interactions of these loci, the Distorters D1,
D2 and D3 act strongly and harmfully on the wild type allele of the
Responder and weakly on the t form of the Responder (R.sup.t),
leading to distortion in favor of R.sup.t. R.sup.t might protect
sperm carrying it from this harmful action of the Distorters. The
Distorters act in trans while the Responder acts in cis. This means
that the chromosome which contains R.sup.t is transmitted at
non-mendelian ratio to the offspring. If D2 or all the Distorters
are present, the chromosome containing R.sup.t is transmitted at a
frequency of more than 50% up to 99% to the offspring. If no
Distorter or only D1 or D3 are present, however, the chromosome
containing R.sup.t is transmitted at less than 50% to the offspring
("low" phenotype). The Distorters are only transmitted at ratios
over 50% if they are located on the same chromosome as is R.sup.t.
The cis-action of R.sup.t suggests that R.sup.t may be expressed at
a stage of spermiogenesis when spermatids are no longer connected
in a syncytium (Willison and Ashworth 1987). This would ensure that
the product of R.sup.t would be restricted to the spermatozoon
containing the t-haplotype form of the R locus. It is expected that
expression in elongating spermatids or mature spermatozoa is
compatible with this requirement. The trans-acting and cis-acting
properties of the Distorters and the Responder, respectively, have
been demonstrated by the transmission ratio properties of so-called
partial t-haplotypes which carry only a subset of the above named
loci (FIG. 1).
[0004] Genetic mapping of molecular markers on partial t-haplotypes
allowed a more or less precise localization of D1, D2, D3 and
R.sup.t to subregions of the T/t-complex and relative to these
molecular markers (Lyon 1984; Fox et al. 1985; Herrmann et al.
1986; Silver and Remis 1987; Bullard et al. 1992). Only one locus,
R.sup.t could be mapped fairly precisely to a region of appr. 200
kb, the so-called T66B region (Fox et al. 1985; Schimenti et al.
1987; Nadeau et al. 1989; Rosen et al. 1990; Bullard et al. 1992).
The T66B region represents a chromosomal piece of the t-haplotype
identified by a t-specific restriction fragment length polymorphism
detected with the probe Tu66 (Fox et al. 1985). The T66B region is
not present in the partial t-haplotypes t.sup.h44 and t.sup.h51,
but is present in the partial t-haplotypes t.sup.low, t.sup.h2,
t.sup.h49, t.sup.6, and in the complete t-haplotypes, e.g. t.sup.w5
or t.sup.w12 (FIG. 1). Another partial t-haplotype, t.sup.w71Jr1
(abbr. t.sup.Jr1) contains T66A and a part of T66B. The chromosomes
t.sup.h44, t.sup.h51 and t.sup.Jr1 do not contain the R.sup.t
function, whereas the other partial and complete t-haplotypes named
above do. The t-haplotypes containing R.sup.t function must have
the t-form of R, whereas the haplotypes t.sup.h44, t.sup.h51 and
t.sup.Jr1 are expected to have the wild type form. The genomic
region T66B has been cloned molecularly and analyzed. A partial
restriction map covering appr. 145 kb of it has been published
(Schimenti et al. 1987; Rosen et al. 1990; Bullard et al.
1992).
[0005] An extensive and careful search of this region for genes
expressed during spermatogenesis has yielded a single gene, T66B-a
or Tcp-10b.sup.t (Schimenti et al. 1988). Further mapping studies
localized "sequences responsible for differential responder
activity" to an interval of 40 kb at the telomeric end of the T66B
region which includes Tcp-10b.sup.t (Bullard et al. 1992). No other
transcription unit could be identified by these authors in the T66B
region within the last 10 years. Tcp-10b.sup.t has been claimed to
represent the candidate for R.sup.t, but a careful analysis showed
that it does not encode Responder properties (Schimenti et al.
1988; Cebra-Thomas et al. 1991; Bullard and Schimenti 1990; Ewulonu
et al. 1996).
[0006] The combined teachings of the prior art thus did not provide
any clue how the genetic elements responsible for the Responder
phenomenon might be identified. More importantly, the analyses
referred to above questioned the prior art discussions that the
Responder is a transcription unit. Accordingly, they taught away
from the possibility that a transcription unit encoding the
Responder might be located in the T66B region. The technical
problem underlying the present invention was, accordingly, to
overcome these long standing prior art difficulties and provide a
genetic entity encoding the Responder function.
[0007] The solution to said technical problem is achieved by
providing the embodiments characterized in the claims.
[0008] Accordingly, the present invention relates to a nucleic acid
molecule comprising a transcription unit encoding in its 5' portion
a kinase having a homology to the MARK2 kinase (Drewes et al.,
1997) as well as to other kinases whereas the 3' portion of the
nucleotide sequence has a high homology to the rsk3 kinase (Zhao et
al., 1995) as well as to expression products thereof. The term
"homology" as used in accordance with the present invention relates
to more than 25% and preferably about 38% identity on the amino
acid level. Thus, in accordance with the present invention, 38%
identity was found in a region of 291 amino acids between MARK2 and
the protein encoded by the nucleic acid molecule shown in FIG. 4a
or 9. Preferably, the kinase gene encoded by the 5' portion lacks
its 3' end which is preferably an untranslated region whereas the
kinase gene encoded by the 3' portion lacks the 5' end and is
preferably not translated.
[0009] Preferably or alternatively, the present invention relates
to a nucleic acid molecule encoding an expression product involved
in the Responder phenotype, which contributes to the phenomenon of
transmission ratio distortion, selected from the group consisting
of
[0010] (a) a nucleic acid molecule comprising the nucleic acid
molecule as shown in FIG. 4a or 9, 7a, 7b, 7c, 7d or a fragment
thereof;
[0011] (b) a nucleic acid molecule being an allelic variant or a
homologue of the nucleic acid sequence of (a);
[0012] (c) a nucleic acid molecule hybridizing to a nucleic acid
molecule complementary to a nucleic acid molecule of (a) or (b);
and
[0013] (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.
[0014] The term "Responder" or "R" as used in accordance with the
present invention relates to mutant as well as wild type forms of
the Responder locus.
[0015] The term "involved in the Responder phenotype", in
accordance with the present invention relates to the fact that
transcripts of all genes displayed on FIG. 4a or 9, 7a, 7b, 7d and
the antisense transcript of 7c are detected in testis carrying
complete t-haplotypes, whereas mapping of the genes displayed on
FIG. 4a or 9 and 7a to the t-Responder region suggests that gene 4a
or 9 and/or 7a is (are) the one(s) encoding t-Responder activity.
In accordance with the further biological data described in this
specification, in particular the data relating to the transgenic
animals, it is proposed that pursuant to this invention, the gene
displayed in FIG. 4a or 9 encodes t-Responder activity. The overall
data suggest that several genes of the Responder (T66Bk) gene
family may act in parallel within t-haplotype carrying spermatids
and/or spermatozoa and are thus presumed to be involved in the
Responder phenotype, whereby it is envisaged that t-Responder
products may antagonize the negative effect of t-Distorter genes
and antisense transcripts derived from Responder genes may reduce
the activity of Responder genes encoding products with t-Responder
as well as wild type or nearly wild-type Responder activity. The
latter products may permit the negative action of t-Distorter
genes.
[0016] It is, furthermore, envisaged in accordance with the present
invention that alternative translation products from one
mRNA-transcript may also be involved in the Responder phenotype
(see, e.g., FIG. 13).
[0017] Specifically the cDNA sequence of T66Bk shown in FIG. 4a or
9 contains the MARK kinase and the rsk3 kinase homology regions.
The cDNA sequence of T66Bk-2 shown in FIG. 7a contains only the
MARK kinase homology region. The cDNA sequence of T66k-8 shown in
FIG. 7b contains the complete sequence of T66Bk-2 except for a
single base deleted between nucleotide position 1508 and 1509
resulting in a frame shift. The cDNA sequence of T66k-7as shown in
FIG. 7c corresponds to an antisense transcript of a T66Bk family
member. The cDNA sequence of T66k-20 shown in FIG. 7d shows a
strong similarity to the above members of the T66Bk gene
family.
[0018] The term "fragment" as used in connection with the nucleic
acid molecule of the invention relates to a fragment that retains
the Responder function. Preferably, said fragment comprises the
portion of the nucleic acid molecule that has a homology to the
MARK kinase referred to above or a part thereof.
[0019] As has been indicated above, in one embodiment of the
nucleic acid molecule of the invention said expression product is
an antisense RNA.
[0020] The term "an allelic variant or a homologue" comprises forms
of the wild type or t-allele of the Responder "gene" which have
been manipulated in vitro in order to achieve an optimal
transmission ratio distortion effect and/or to adapt it to the
specific requirements of the breeding scheme employed, thus
improving the selectability of genetic traits. A number of standard
manipulations known in the field are taken into consideration, such
as those resulting in the exchange of phosphorylation sites (Ser,
Thr, Tyr) on the Responder (poly)peptide for acidic or neutral
(Ala) amino acid residues, mutagenesis of the active center,
overexpression or knock out mutagenesis of said gene, construction
of hypomorphic (poly)peptides by mutagenesis of ATP and/or GTP
binding site(s), deletion of phosphorylation sites on said
(poly)peptide, deletion of binding sites for other (poly)peptides
involved in the Responder/Distorter signaling cascade, synthesis of
antisense RNA, N-terminal or C-terminal truncations, introduction
of frame shifts which alter part of the amino acid sequence of the
protein, etc., resulting either in null, hypomorphic,
constitutively active, antimorphic or dominant negative alleles. It
is also envisaged that a distortion of the transmission ratio can
be achieved with several, if not all, manipulated forms of the
Responder gene suggested above. Thus, a manipulated Responder
allele affecting the transmission ratio most effectively will have
to be identified empirically by employing activity assays in cell
culture systems and by employing transgenic animal systems.
[0021] It is also envisaged that one or several members of the
T66Bk kinase gene family might function as Distorter(s), provided
it is (they are) expressed during the diploid or early haploid
phase of spermatogenesis allowing distribution of the gene products
to all spermatozoa, or can be altered in vitro such as to function
as Distorters. The latter may be achieved by in vitro manipulations
such as those resulting in the exchange of phosphorylation sites
(Ser, Thr, Tyr) on said Responder (poly)peptide for acidic or
neutral (Ala) amino acid residues, N- or C-terminal truncation,
frame shift, deletion of phosphorylation sites, deletion of binding
sites for other (poly)peptides, mutagenesis of the active center,
or overexpression of said gene or of antisense transcripts,
resulting in constitutively active, hypomorphic, antimorphic or
dominant negative gene products and expression of said gene
products during the diploid or early haploid phase of
spermatogenesis allowing distribution of the gene products to all
spermatozoa, e.g. under the control of the Pgk2 promoter. These
manipulations are envisaged to have a negative effect on sperm
motility and/or fertilization capability. This negative effect may
then be balanced by Responder constructs having the opposite
effect. The latter could be restricted to those spermatozoa
carrying the construct by expressing it under the control of the
Responder gene promoter. It is envisaged that both types of
spermatozoa would be negatively affected by the Distorter construct
expressed in the diploid phase of spermatogenesis, whereas the
sperm carrying, in addition, the Responder construct expressed in
spermiogenesis would be partially or completely protected by the
(poly)peptide expressed in it, and would thus gain an advantage in
sperm motility and/or fertilization capability over the other
sperm. This would lead to a transmission ratio distortion in favor
of the "protected" spermatozoa. Preferably the Distorter construct
expressed in both types of spermatozoa would encode a hypermorphic
or constitutively active (poly)peptide, whereas the Responder
construct expressed only in those spermatozoa carrying it should
encode a hypomorphic, antimorphic or dominant negative
(poly)peptide. Both constructs could be integrated on the same or
on different chromosomes. Preferably both constructs would be
integrated together on the X- or the Y-chromosome, resulting in the
preferential or exclusive transmission of the X- or Y-chromosome
and thus the preferential or exclusive fathering of female or male
offspring, respectively.
[0022] The term "hybridizing" as used in connection with the
present invention relates to stringent or nonstringent
hybridization conditions. Preferably, it relates to stringent
conditions. Said hybridization conditions may be established
according to conventional protocols described, for example, in
Sambrook, "Molecular Cloning, A Laboratory Manual", Cold Spring
Harbor Laboratory (1989) N.Y., Ausubel, "Current Protocols in
Molecular Biology", Green Publishing Associates and Wiley
Interscience, N.Y. (1989), or Higgins and Hames (eds) "Nucleic acid
hybridization, a practical approach" IRL Press oxford, Washington
D.C., (1985). Stringent hybridization conditions are, for example,
hybridization in 6.times.SSC, 5.times. Denhardt's reagent, 0.5%
SDS, and 100 .mu.g/ml denatured DNA at 65.degree. C. and washing in
0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0023] In accordance with the present invention and in contrast to
the teachings of the prior art, it was surprisingly found that
nucleic acid sequences responsible for the Responder phenotype are
comprised at the centromere-close part of the T66 B region. It
conforms with several criteria that would be expected for the
Responder function:
[0024] a) it is located in the T66B region;
[0025] b) it is expressed in testis; and
[0026] c) it is expressed during spermatogenesis.
[0027] In accordance with the present invention, it is further
envisaged that additional expression products may contribute to
Responder function as has been indicated above which are not
necessarily located in the B-region.
[0028] As has been indicated above, one of the transcription units
(namely T66Bk) contributing to the Responder (R) phenotype
apparently arises from two truncated genes. One of said genes has a
high homology to the rsk3 gene, the second one has an homology to
the MARK kinase recently identified (Drewes et al., 1997). Another
transcription unit envisaged to contribute to the R phenotype,
T66Bk-2, also has a homology to the MARK kinase, but lacks homology
to the rsk3 gene as indicated above. The identification of the
genetic basis underlying the R phenotype allows a number of genetic
manipulations, in particular in connection with breeding schemes,
to be conveniently carried out in the future. Such schemes will be
addressed in more detail herein below.
[0029] In accordance with the present invention, it is envisaged
that the expression products encoded by the nucleic acid sequences
of the invention may contribute to the Responder phenotype in
several different ways. Thus, in one embodiment one of the above
indicated expression products are themselves sufficient to distort
the transmission ratio. In another embodiment all of said
expression products or combinations of them have to be provided in
order to distort the transmission ratio, with certain combinations
being more effective than others. In yet another embodiment of the
present invention said expression products may work in an additive
or synergistic manner. In a still further embodiment it is
envisaged that antisense transcripts derived from one or several
genes of the T66Bk gene family may contribute to the t-Responder
function resulting in a lower level or abolishment of mRNA of one
or several T66Bk genes and thus a lower level or abolishment of the
corresponding (poly)peptides translated from said mRNA molecules.
An example of such an antisense transcript is shown in FIG. 7c.
Furthermore, it is suggested that the specifically identified
nucleic acid sequences coding for expression products involved in
the R phenotype may not be the only ones responsible for the
Responder phenotype. Thus, it is envisaged that further nucleic
acids encoding expression products that act in concert with the
ones discussed above and that may contribute to the Responder
phenotype are contained in the genome. Additionally, it is
envisaged in accordance with the present invention that the nucleic
acid molecules of the invention exert or enhance the Responder
phenotype in conjunction with further sequences comprised, for
example, in the T66A, T66B and T66C regions. Preferably, said
additional regions encode MARK-related kinases.
[0030] Also, the person skilled in the art will, on the basis of
the teachings of the present invention, be in a position to
genetically manipulate the nucleic acid contributing to the
Responder phenotype. He will further be in the position to screen
the genome of an organism or cell of interest for additional
nucleic acid sequences encoding Responder functions on the basis of
the genetic organization of the Responder taught in accordance with
the present invention. All these embodiments that are without
further ado derivable from the specific teachings provided herein
are also comprised by the present invention.
[0031] It is further envisaged in accordance with the present
invention that the Responder may act as a component of a signaling
cascade involved in sperm motility and/or the fertilization of
oocytes. The t-Responder may act such as to protect the sperm
carrying the t-form of the Responder from the negative actions of
the t-Distorters whereas the sperm carrying the wild type form of
the Responder is "poisoned" (see Lyon 1986). Therefore, the action
of the t-form of the Responder somehow counteracts the t-Distorter
function suggesting that the Distorters are part of the same
signaling cascade. It is, thus, envisaged that the wild type gene
or the products of any member of that signaling cascade, once
molecularly known, can be manipulated such as to "poison" the sperm
expressing either dominant active or dominant negative forms, or by
overexpressing, reducing or abolishing the gene function of any
member of said signaling cascade. Selection of genetic traits may
then be easily achieved by manipulating the amino acid sequence,
activity or expression level of any member of that signaling
cascade and restricting the expression of the manipulated form
preferentially or completely to those sperm carrying it, such as is
the case for the Responder function. The promoter of the Responder
or other promoters activating gene expression during the haploid
phase of spermatogenesis would be a suitable means for achieving
this restriction.
[0032] Accordingly, the present invention also relates to methods
of influencing transmission ratio by manipulating the expression
level or the protein activity of any other member of said signaling
cascade. For the purposes of this invention, said cascade is termed
"Responder/Distorter signal cascade". It is further envisaged in
accordance with the present invention that other signaling cascades
may exist besides the Responder/Distorter signaling cascade that
may be involved in the motility and/or fertilization capability of
spermatozoa. Thus, it is envisaged in accordance with the present
invention that the expression level and/or activity of one or more
of the proteins involved in said other signaling cascades may be
also manipulated in order to influence the transmission ratio.
Influencing transmission ratio implies that said ratio may be
enhanced or reduced. Methods for manipulating said expression level
or said protein activity are known in the art and comprise methods
of manipulating amino acid sequences and/or, e.g., promoter
strengths or expressing an inhibitor of any member of said
signaling cascade. Alternatively, it is envisaged that the
expression level may be modulated on the transcription level, the
level of pre-mRNA processing, mRNA transport and/or stability,
and/or the translation level. Preferably, the modification and/or
replacement of elements does not alter the tissue specificity or
the specificity for the developmental stage of the expression unit.
It is also envisaged in accordance with the present invention that
the genetic background of the host organism, the site of
integration, and/or the number of integrated copies of a transgene
construct may influence the expression efficiency of said transgene
construct. Expression or activity of one or more of said members
may (significantly) be altered or enhanced, (significantly) be
reduced or abolished. Said members also include the Distorters.
These methods of the invention can, either alone or in conjunction
with other methods described below, advantageously be used for the
generation of transgenic animals. Said transgenic animals provide a
suitable assay system to test whether the above mentioned methods
for manipulating said expression level or said protein activity
were successful. Such a system is described in Example 6.
Furthermore, said transgenic animals may be employed in any of the
breeding schemes addressed below.
[0033] In another preferred embodiment of the invention, said
nucleic acid molecule is a DNA molecule.
[0034] The deduction of the amino acid sequence from the nucleic
acid sequence of the invention allows the conclusion that the
polypeptide is the expression product that contributes to the
Responder phenotype. However, it is not excluded that the mRNA
contributes to or triggers said Responder phenotype. Also, it is
envisaged in accordance with the present invention that the
expression level, stage of expression during spermatogenesis or the
copy number of said gene results in or contributes to the Responder
phenotype. Therefore, in a preferred embodiment of the nucleic acid
molecule of the invention said expression product is an RNA or a
(poly)peptide.
[0035] A further preferred embodiment of the invention is a nucleic
acid molecule, wherein said Responder function is the
mouse-t-complex Responder function. Although it is easily possible
to identify mutated or wild-type Responders in animals other than
the mouse on the basis of the genetic structure of the Responder
that is provided in accordance with the present invention, the
mouse t-complex Responder may find applications, for example in
breeding, also when introduced into other animals. Specific
applications of the Responder function are addressed herein
below.
[0036] The invention further relates to a regulatory region of the
gene corresponding to the nucleic acid molecule of the invention
being capable of controlling expression of said nucleic acid
molecule.
[0037] The term "corresponding" as used in accordance with the
present invention also means that the gene comprises the nucleic
acid molecule of the invention or fragments thereof.
[0038] The term "regulatory region" in the present application
refers to sequences which influence the specificity and/or level of
expression, for example in the sense that they confer cell and/or
tissue specificity. Such regions can be located upstream of the
transcription initiation site, but can also be located downstream
of it, e.g., in transcribed leader sequences or in an intron.
[0039] The term "a regulatory region of the gene corresponding to
the nucleic acid molecule" refers to a region with the above
mentioned capabilities that controls expression of the bipartite
nucleic acid molecule referred to herein also as a "gene".
[0040] Regulatory elements ensuring expression in eukaryotic cells,
preferably mammalian cells, are well known to those skilled in the
art. They usually comprise promoters ensuring initiation of
transcription and optionally poly-A signals ensuring termination of
transcription and stabilization of the transcript. Additional
regulatory elements may include transcriptional as well as
translational enhancers.
[0041] Preferably, said regulatory region is a naturally occurring
regulatory region or a genetically engineered derivative
thereof.
[0042] More preferably, said regulatory region comprises or is a
promoter. Said promoter is preferably tissue specific and confers
expression, for example, during spermiogenesis.
[0043] The term "promoter" refers to the nucleotide sequences
necessary for transcription initiation, i.e. RNA polymerase
binding, and also includes, for example, the TATA box.
[0044] In one embodiment, said promoter is or comprises a minimal
promoter.
[0045] According to the present invention, promoters from other
species can be used that are functionally homologous to the
regulatory sequences or the promoter of the murine gene, or
promoters of genes that display an identical pattern of expression,
in the sense of being expressed in sperm cells. As has been
outlined above, it is possible for the person skilled in the art to
isolate with the help of the known murine nucleic acid
corresponding genes from other species, for example, human. This
can be done by conventional techniques known in the art, for
example, by using the nucleic acid molecule of the invention as a
hybridization probe or by designing appropriate PCR primers. It is
then possible to isolate the corresponding promoter region by
conventional techniques and test it for its expression pattern. For
this purpose, it is, for instance, possible to fuse the promoter to
a reporter gene, such as the lacZ gene or green fluorescent protein
(GFP) and assess the expression of the reporter gene in transgenic
mice.
[0046] The present invention also relates to the use of promoter
regions which are substantially identical to the murine promoter or
to a promoter of a homologous gene or to parts thereof and which
are able to confer specific expression in sperm cells.
[0047] Such promoters differ at one or more positions from the
above-mentioned promoters but still have the same specificity,
namely they comprise the same or similar sequence motifs
responsible for the above described expression pattern. Preferably
such promoters hybridize to one of the above-mentioned promoters,
most preferably under stringent conditions. Particularly preferred
are promoters which share at least 85%, more preferably 90-95%, and
most preferably 96-99% sequence identity with one of the
above-mentioned promoters and have the same specificity. Such
promoters also comprise those which are altered, for example by
deletion(s), insertion(s), substitution(s), addition(s), and/or
recombination(s) and/or any other modification(s) known in the art
either alone or in combination in comparison to the above-described
nucleotide sequence. Methods for introducing such modifications in
the nucleotide sequence of the promoter of the invention are well
known to the person skilled in the art. It is also immediately
evident to the person skilled in the art that further regulatory
sequences may be added to the promoter of the invention. For
example, transcriptional enhancers and/or sequences which allow for
induced expression of the promoter of the invention may be
employed. A suitable inducible system is for example
tetracycline-regulated gene expression as described, e.g., by
Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551)
and Gossen et al. (Trends Biotech. 12 (1994), 58-62).
[0048] Most preferably, said regulatory region comprises the
fragment from nucleotides 930 to 3576 of the sequence shown in FIG.
11.
[0049] Also comprised are fragments or variants of the above
sequence wherein the regulatory function of said fragments or
variants is essentially retained or even improved. This may be
tested according to methods well known in the art in combination
with the teaching of this specification.
[0050] The invention further relates to a recombinant DNA molecule
comprising a nucleic acid molecule of the invention and/or a
regulatory region of the invention and/or a regulatory region
allowing expression during spermatogenesis/ spermiogenesis.
[0051] Accordingly, the regulatory region may control expression of
the nucleic acid molecule contributing to the Responder function.
Alternatively, said recombinant DNA molecule may comprise said
regulatory region which controls expression of a heterologous
nucleic acid or which is not operatively linked to any nucleic acid
and, thus, may be used for cloning purposes. In the first
alternative, said regulatory region is operatively linked to a
heterologous DNA sequence. For example, said regulatory region may
be operatively linked to a naturally occurring or in vitro
engineered DNA encoding a member of the Responder/Distorter
cascade, for example, a Distorter or a member of another signaling
cascade involved in sperm motility and/or fertilization. Also, in
this embodiment of the invention, the nucleic acid molecule of the
invention may be operatively linked to a different or to no
regulatory region. The regulatory region may be the original
regulatory region of the gene corresponding to the nucleic acid
molecule of the invention or may be derived from a different copy
of said gene or from a different gene. Furthermore, the regulatory
region may be derived from a copy of the homologous gene (in case
more than one copy exists) from a different species or may be
derived from a different gene from said different species. The
above mentioned regulatory regions may also be modified in order to
obtain optimum expression, which may be enhanced or reduced
expression. Thus, it is envisaged in accordance with the present
invention that e.g., the regulatory regions controlling expression
of the gene comprising the T66k-20-cDNA (see FIG. 7d) or the cDNAs
shown in FIG. 10 are used in unmodified or modified form in
accordance with the present invention. Due to the teaching of the
present invention, namely the cloning and the disclosure of the
sequences of the cDNAs, it is routine experimentation for the
person skilled in the art to clone and use said regulatory
regions.
[0052] Advantageously, the recombinant DNA molecule of the
invention may further comprise an expression unit encoding and
expressing a desired genetic trait. Such a DNA molecule may be used
to reduce, or enhance the inheritance of said desired genetic
trait, provided that either the recombinant DNA molecule further
comprises an expression unit encoding and expressing at least one
Distorter or protein with Distorter activity, preferably D2, or the
genetic background of the host provides such Distorter activity
which may be naturally occurring in said host or which may have
been introduced.
[0053] A particularly preferred embodiment of the invention relates
to a recombinant DNA molecule, wherein said heterologous DNA
sequence encodes a peptide, protein, antisense RNA, sense RNA
and/or ribozyme.
[0054] As regards the antisense RNA, it may find applications in
methods of antisense therapy or antisense knockout strategies.
Antisense therapy may be carried out by administering to an animal
or a human patient, a recombinant DNA containing the regulatory
sequences of the invention operably linked to a DNA sequence, i.e.,
an antisense template which is transcribed into an antisense RNA.
The antisense RNA may be a short (generally at least 10, preferably
at least 14 nucleotides, and optionally up to 100 or more
nucleotides) nucleotide sequence formulated to be complementary to
a portion of a specific mRNA sequence. Standard methods relating to
antisense technology have been described (Melani, Cancer Res. 51
(1991), 2897-2901). Following transcription of the DNA sequence
into antisense RNA, the antisense RNA binds to its target mRNA
molecules within a cell, thereby inhibiting translation of the mRNA
and down-regulating expression of the protein expected to be
encoded by the mRNA. For example, an antisense sequence will be
complementary to a portion of or all of the mRNA. In addition,
ribozymes may advantageously be employed to eliminate wild-type
Responder transcripts from cells.
[0055] The invention further relates to a recombinant DNA molecule,
wherein said peptide, protein, antisense RNA, sense RNA, a toxin
and/or ribozyme is capable of causing cell death.
[0056] In this embodiment of the invention, sperm which do not
carry the R related transgene can be genetically selected.
[0057] For example, the promoter of the R gene can be used for the
expression of a gene product inducing the destruction or apoptosis
of said spermatocytes carrying said construct. Integration of such
a construct on the X- or Y-chromosome will result in the
transmission of the respectively other sex chromosome. Integration
of the construct on the X chromosome will lead to the neutral
transmission of the construct in female animals. Integration in the
Y chromosome should, preferably, be in an inactive state that can
be activated along the rules that will be laid down herein
below.
[0058] A recombinant DNA molecule which further comprises DNA
encoding an effector polypeptide is a further preferred embodiment
of the invention.
[0059] It is particularly preferred that said effector polypeptide
is capable of sequestering an ion selectively binding to a solid
support, or binding to a preselected antigenic determinant or is a
toxin, an enzyme, a ribozyme, a label or a remotely detectable
moiety.
[0060] In accordance with the invention, it is most preferred that
said effector polypeptide is calmodulin, methallothionein, a
fragment thereof, green fluorescent protein (GFP), .beta.-lactamase
(Zlokarnik et al., 1998), hCD24, myc, FLAG, hemagglutinin or an
amino acid sequence rich in at least one of glutamic acid, aspartic
acid, lysine, histidine or arginine.
[0061] Accordingly and in other words, the above embodiments of the
invention relate to the use of the R promoter for the expression of
a (poly)peptide being or having a tag. Said tag may be expressed in
the cytoplasm of sperm. An example of such a tag is GFP or
.beta.-lactamase. Said tag is alternatively located on the surface
of sperm and thus, may be recognized by specific antibodies. This
enables the separation of sperm carrying a transgene expressed
under the control of the R promoter from sperm not carrying said
transgene. The person skilled in the art is familiar with a variety
of methods for the separation of sperm carrying said tag on its
surface. Preferably, said tag is selected by affinity
chromatography or by using a cell sorter. After separation, sperm
carrying the transgene or sperm without the transgene can be used
for fertilization of eggs. This embodiment includes integration of
transgene in either autosomes or sex chromosomes.
[0062] Advantageously, the solid support referred to above is a
membrane or the surface of an ELISA plate.
[0063] Further, the invention relates to a vector comprising the
nucleic acid molecule of the invention, the regulatory region of
the invention or the recombinant DNA molecule of the invention.
[0064] The vector of the invention may simply be used for
propagation of the genetic elements comprised therein.
Advantageously, it is an expression vector and/or a targeting
vector. Expression vectors such as Pichia pastoris derived vectors
or vectors derived from viruses such as CMV, SV-40, baculovirus or
retroviruses, vaccinia virus, adeno-associated virus, herpes
viruses, or bovine papilloma virus, may be used for delivery of the
recombinant DNA molecule or vector of the invention into targeted
cell population. Methods which are well known to those skilled in
the art can be used to construct recombinant viral vectors; see,
for example, the techniques described in Sambrook, loc. cit. and
Ausubel, loc. cit. Alternatively, the recombinant DNA molecules and
vectors of the invention can be reconstituted into liposomes for
delivery to target cells.
[0065] It is preferred according to one further embodiment that
said vector comprises a heterologous promoter.
[0066] Said heterologous promoter not naturally operatively linked
with the nucleic acid contributing to the Responder function may be
used to determine a certain time point of the onset of Responder
expression. This time point may be the same or a different one that
is set when the natural Responder transcription unit is employed.
For example, said heterologous promoter may also be active in the
early or late haploid phase of spermatogenesis.
[0067] It is particularly preferred that said heterologous promoter
is controlling gene expression in spermatogenesis and/or in
spermiogenesis.
[0068] Most preferably, said heterologous promoter is the testis
promoter of c-kit or of Angiotensin-Converting-Enzyme (ACE), both
of which are well known in the art.
[0069] The invention further relates to a host cell transformed or
transfected with the nucleic acid molecule, the recombinant DNA
molecule or the vector of the invention.
[0070] The host cell can be any prokaryotic or eukaryotic cell,
such as a bacterial, insect, fungal, plant, animal or human cell.
Prokaryotic host cells will usually only be employed for the
propagation of the nucleic acid molecule of the invention and
sometimes for the production of the expression product. Suitable
mammalian, fish or bird cell lines are well known or can easily be
determined by the person skilled in the art and comprise COS cells,
Hela cells, primary embryonic cell lines etc.
[0071] The term "transfected or transformed" is used herein in its
broadest possible sense and also refers to techniques such as
electroporation, infection or particle bombardment.
[0072] Furthermore, the invention relates to a method of
recombinantly producing an expression product as defined herein
above comprising the steps of culturing the host cell of the
invention under conditions to cause expression of the protein and
recovering said protein from the culture.
[0073] The method of the invention is most advantageously carried
out along conventional protocols which have been described, for
example, in Sambrook, loc. cit.
[0074] The invention further relates to an expression product
encoded by the nucleic acid molecule of the invention or which is
obtainable by the production method of the invention.
[0075] In accordance with the invention, said expression product
may either be an mRNA or a polypeptide. Said expression product is,
in accordance with the present invention, involved in the Responder
phenotype and contributes to the phenomenon of transmission ratio
distortion.
[0076] A further embodiment of the invention relates to an antibody
specifically recognizing the expression product of the
invention.
[0077] The antibody of the invention may be a monoclonal antibody
or an antibody comprised in a polyclonal serum. Accordingly, the
term "antibody" as used herein also relates to a polyclonal
antiserum. In addition, said term relates to antibody fragments or
fusion proteins comprising antibody binding sites such as Fab, Fv,
scFv fragments etc. The antibody of the invention has a number of
applicabilities including purification or diagnostic processes.
[0078] The invention additionally relates to a nucleic acid
molecule specifically hybridizing with the nucleic acid molecule of
the invention translatable into said MARK related kinase or to an
intron of said nucleic acid molecule or with the regulatory region
of the invention or with a complementing strand thereof.
[0079] Said nucleic acid molecules comprise at least 15 nucleotides
in length and hybridize specifically with a nucleic acid or
regulatory sequence as described above or with a complementary
strand thereof. Specific hybridization occurs preferably under
stringent conditions and implies no or very little
cross-hybridization with nucleotide sequences having no or
substantially different regulatory properties. Such nucleic acid
molecules may be used as probes and/or for the control of gene
expression. Nucleic acid probe technology is well known to those
skilled in the art who will readily appreciate that such probes may
vary in length. Preferred are nucleic acid probes of 17 to 35
nucleotides in length. Of course, it may also be appropriate to use
nucleic acids of up to 100 and more nucleotides in length. The
nucleic acid probes of the invention are useful for various
applications. On the one hand, they may be used as PCR primers for
amplification of regulatory sequences according to the invention.
In this embodiment, one of the primers may hybridize to the 3'
portion of the Responder having a high homology to the rsk3 gene.
Another application is the use as a hybridization probe to identify
regulatory sequences hybridizing to the regulatory sequences of the
invention by homology screening of genomic DNA libraries. Nucleic
acid molecules according to this preferred embodiment of the
invention which are complementary to a regulatory sequence as
described above may also be used for repression of expression of a
gene comprising such regulatory sequences, for example due to an
antisense or triple helix effect or for the construction of
appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201,
EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene
comprising a regulatory sequence of the invention. Selection of
appropriate target sites and corresponding ribozymes can be done as
described for example in Steinecke, Ribozymes, Methods in Cell
Biology 50, Galbraith et al. eds Academic Press, Inc. (1995),
449-460. Furthermore, the person skilled in the art is well aware
that it is also possible to label such a nucleic acid probe with an
appropriate marker for specific applications, such as for the
detection of the presence of a nucleic acid molecule of the
invention in a sample derived from an organism.
[0080] The above described nucleic acid molecules may either be DNA
or RNA or a hybrid thereof. Furthermore, said nucleic acid molecule
may contain, for example, thioester bonds and/or nucleotide
analogues, commonly used in oligonucleotide anti-sense approaches.
Said modifications may be useful for the stabilization of the
nucleic acid molecule against endo- and/or exonucleases in the
cell. Said nucleic acid molecules may be transcribed by an
appropriate vector containing a chimeric gene which allows for the
transcription of said nucleic acid molecule in the cell. Such
nucleic acid molecules may further contain ribozyme sequences which
specifically cleave the (pre)-mRNA comprising the regulatory
sequence of the invention. Furthermore, oligonucleotides can be
designed which are complementary to a regulatory sequence of the
invention (triple helix; see Lee, Nucl. Acids Res. 6 (1979), 3073;
Cooney, Science 241 (1988), 456 and Dervan, Science 251 (1991),
1360), thereby preventing transcription and the production of the
encoded mRNA and/or protein.
[0081] Furthermore, the invention relates to a pharmaceutical
composition comprising the DNA molecule, the regulatory region, the
recombinant DNA, the vector, the host cell, the expression product
or the antibody of the invention.
[0082] Said pharmaceutical composition comprises at least one of
the aforementioned compounds of the invention, either alone or in
combination, and optionally a pharmaceutically acceptable carrier
or excipient. Examples of suitable pharmaceutical carriers are well
known in the art and include phosphate buffered saline solutions,
water, emulsions, such as oil/water emulsions, various types of
wetting agents, sterile solutions etc. Compositions comprising such
carriers can be formulated by conventional methods. These
pharmaceutical compositions can be administered to subject in need
thereof at a suitable dose. Administration of the suitable
compositions may be effected by different ways, e.g., by
intravenous, intraperitoneal, subcutaneous, intramuscular, topical,
intradermal, intranasal or intrabronchial administration. The
dosage regimen will be determined by the attending physician and
other clinical factors. As is well known in the medical arts,
dosages for any one patient depends upon many factors, including
the patient's size, body surface area, age, the particular compound
to be administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. A typical
dose can be, for example, in the range of 0.001 to 1000 .mu.g (or
of nucleic acid for expression or for inhibition of expression in
this range); however, doses below or above this exemplary range are
envisioned, especially considering the aforementioned factors.
Generally, the regimen as a regular administration of the
pharmaceutical composition should be in the range of 1 .mu.g to 10
mg units per day. If the regimen is a continuous infusion, it
should also be in the range of 1 .mu.g to 10 mg units per kilogram
of body weight per minute, respectively. Progress can be monitored
by periodic assessment. Dosages will vary but a preferred dosage
for intravenous administration of DNA is from approximately
10.sup.6 to 10.sup.22 copies of the nucleic acid molecule. The
compositions of the invention may be administered locally or
systematically. Administration will generally be parenterally,
e.g., intravenously; DNA may also be administered directly to the
target site, e.g., by biolistic delivery to an internal or external
target site or by catheter to a site in an artery. Preparations for
parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's,
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like.
Furthermore, the pharmaceutical composition of the invention may
comprise further agents such as interleukins or interferons
depending on the intended use of the pharmaceutical
composition.
[0083] It is envisaged by the present invention that in particular
the various recombinant nucleic acid/DNA molecules and vectors of
the invention are administered either alone or in any combination
using standard vectors and/or gene delivery systems, and optionally
together with an appropriate compound and/or together with a
pharmaceutically acceptable carrier or excipient. Subsequent to
administration, said molecules may be stably integrated into the
genome of the mammal, fish or bird. On the other hand, viral
vectors may be used which are specific for certain cells or
tissues, preferably for pancreatic cells and persist in said cells.
Suitable pharmaceutical carriers and excipients are well known in
the art.
[0084] The invention further relates to a diagnostic composition
comprising the nucleic acid molecule, the regulatory region, the
recombinant DNA molecule, the vector, the host cell, the expression
product or a primer or an oligonucleotide hybridizing to the
nucleic acid molecule or regulatory region of the invention or to a
complementary strand thereof and preferably to the regions
identified herein above or the antibody of the invention. Comprised
by the above definition of the term "primer" are also pairs of
primers such as forward and reverse primers that may be used for
PCR. One of said primers of said pair of primers may hybridize in
the region of the rsk-related nucleic acid sequence.
[0085] In one embodiment, said diagnostic composition is
manufactured in the form of a kit. Said compositions may
additionally contain further compounds such as plasmids,
antibiotics and the like for screening animals or cells for the
presence of nucleic acid sequences or regulatory elements
corresponding to those identified in the appended examples or
described herein above.
[0086] The components of the diagnostic composition and/or kit of
the present invention may be packaged in containers such as vials,
optionally in buffers and/or solutions. If appropriate, one or more
of said components may be packaged in one and the same container.
Additionally or alternatively, one or more of said components may
be adsorbed to a solid support such as, e.g., a nitrocellulose
filter or nylon membrane, or to the well of a microtiter plate.
[0087] The invention further relates to a method for the production
of a transgenic non human mammal, fish or bird comprising
introducing the nucleic acid molecule, the regulatory region, the
recombinant DNA molecule or the vector of the invention into a
cell, preferably germ cell, embryonic cell or an egg cell or a cell
derived therefrom.
[0088] Methods for the generation of such transgenic animals are
well known in the art and are described, for example, in "Guide to
techniques in mouse development" (ed. Wassarman & DePamphilis)
Methods in Enzymology Vol. 225 (Academic Press, 1993). The method
of the invention also comprises embodiments related to the cloning
of such animals. These embodiments include the steps of introducing
said nucleic acid molecule, recombinant DNA molecule or vector of
the invention into the nucleus of a cell, preferably an embryonic
cell, replacing the nucleus of an oocyte, a zygote or an early
embryo with said nucleus comprising said nucleic acid molecule,
recombinant DNA molecule or vector of the invention, transferring
either said ooyte, zygote or early embryo into a foster mother or
first in vitro or in vivo culturing said oocyte, zygote or early
embryo and subsequently transferring the resulting embryo into a
foster mother and allowing the embryo to develop to term; see, for
example, Wilmut I. et al. (1997) "Viable offspring derived from
fetal and adult mammalian cells", Nature 385, 810-813.
[0089] In a preferred embodiment of the method of the invention,
said chromosome is an X chromosome or the corresponding sex
chromosome in birds or fish or an autosome.
[0090] In an alternative preferred embodiment of the method of the
invention, said chromosome is a Y chromosome, or the corresponding
sex chromosome in birds or fish.
[0091] It is particularly preferred that the nucleic acid molecule,
the regulatory region, the recombinant DNA molecule or the vector
of the invention, a heterologous promoter controlling expression in
spermiogenesis and/or a DNA sequence encoding an effector
(poly)peptide as defined hereinabove alone or in combination is/are
integrated in said Y chromosome in a reversible inactive state of
expressibility.
[0092] In accordance with the method of the invention, it is most
preferred that said nucleic acid molecule, regulatory region,
recombinant DNA molecule, vector of the invention, a heterologous
promoter controlling expression in spermiogenesis and/or a DNA
sequence encoding an effector (poly)peptide as defined hereinabove
alone or in combination is/are flanked by lox P sites or FRT
sites.
[0093] In all the above embodiments, at least one Distorter may be
present on the same or on different chromosome.
[0094] An additional particularly preferred embodiment of the
method of the invention further comprises 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.
[0095] Advantageously, said Distorters are the mouse t-complex
Distorter loci.
[0096] It is most preferred that said Distorter is/are D2 and/or
D1.
[0097] Said method of the invention and its various preferred
embodiments provide a wide range of applications in particular in
the breeding of animals. Thus, as has been outlined above, the
nucleic acid sequence encoding a molecule contributing to the
Responder and/or an effector (poly)peptide as defined hereinabove
may be under the regulation of the promoter naturally associated
with said nucleic acid sequence. Integration of such a construct
into a chromosome will, in the absence of a Distorter function
result in a disadvantage in a chromosome if it comes to
transmission of said chromosome. This disadvantage may be in the
range of 49 to 0% transmission ratio. In the case that the
Responder effect results in a very low or no transmission of the
corresponding chromosome and if, in addition, the above recited
construct comprising the nucleic acid molecule of the invention or
the effector (poly)peptide is integrated into the Y chromosomes,
the Y chromosome and the Responder function would hardly or not be
transmitted by male animals. In order to provide for male animals,
the Y chromosome should advantageously comprise an inactive
construct that can, however, be activated. Said inactive construct
should be without influence on the transmission ratio. One
embodiment of said construct comprises loxP or FRT sites which
flank an intervening sequence located between said promoter or a
heterologous promoter controlling expression in spermiogenesis and
effector (poly)peptide encoding sequences and/or sequences
conferring Responder activity. The intervening sequence would be
designed in such a way as to prevent the expression of effector
and/or Responder activity. Activation of the effector and/or
Responder activity may be effected by excision of the intervening
sequence due to activity of the Cre or flp protein comprised in the
same cell. Another embodiment of said construct comprises loxP or
FRT sites flanking said promoter or a heterologous promoter
controlling expression in spermiogenesis whereby the promoter is
oriented away from the construct comprising the nucleic acid of the
invention or the effector sequences encoding the above mentioned
(poly)peptides. The activity of Cre or flp would allow the promoter
to be inverted resulting in the transcription of the effector
sequences or the sequences contributing to Responder activity
during spermiogenesis. Another embodiment of said construct
comprises loxP or FRT sites flanking said nucleic acid sequences
reversely oriented towards the promoter such that the antisense
strand is transcribed during spermiogenesis. Activation may be
effected by flipping the effector sequences or the sequences
contributing to Responder activity due to the activity of Cre or
flp comprised in the same cell. Expression of the Cre or flp
protein would advantageously be effected prior to spermiogenesis.
The activation of the Responder or effector function is in such
cases effected during spermatogenesis under the control of the R
promoter or another promoter controlling expression during
spermatogenesis/spermiogenesis. Preferably, the Cre gene is
integrated on an autosome and may be expressed under the control of
one of the following promoters: cytomegalovirus immediate early
enhancer-chicken beta-actin hybrid (CAG) promoter, wherein site
specific recombination occurs in the zygote; adenovirus Ella
promoter, wherein expression is triggered during early
embryogenesis; CMV, wherein expression is triggered during
embryogenesis; OCT4, wherein expression is also triggered during
embryogenesis and in germ line cells; HSV-TK or Pgk, wherein
expression is ubiquitous; or Pgk2, wherein the construct is
expressed during spermatogenesis. In the above embodiment, the
Responder and/or effector encoding construct is transmitted by male
animals in an inactive state. Mating with a female carrier of the
Cre construct will result in male progeny having their Responder
and/or effector activated during spermatogenesis. Progeny of these
male animals inherit predominantly or exclusively the X chromosome
of the father and are accordingly female progeny. In the case that
the X chromosome is exclusively transmitted, the Responder and/or
effector function is not inherited by the progeny. However, in
cases of a less strong effect of the Responder and/or effector
(poly)peptide leading to, for example, 10 to 20% transmission, the
inactivation of the construct is not necessary because this low
transmission is sufficient for the generation of male carriers. The
frequency of inheritance of the R gene of the mouse, without the
interaction of t-Distorters, is naturally in the range of about
20%.
[0098] In an alternative preferred embodiment of the method of the
invention that has been identified above, the Responder and/or
effector is integrated on the X chromosome or on an autosome. In
this case, no inactive construct is necessary, since the Responder
and/or effector encoding construct is transmitted in female animals
in a neutral state, because Responder function only acts during
spermatogenesis. Mating with wild type male animals leads to the
generation of male animals carrying an active R and/or effector
encoding gene on the X chromosome or an autosome. The chromosome
carrying the R and/or effector encoding gene has a disadvantage in
transmission. This means less than 50% to 0% of the progeny inherit
said chromosome. In the case that the R and/or effector encoding
construct is integrated into the X chromosome, no female progeny or
only a low percentage of female progeny will be generated.
[0099] Furthermore, the invention relates to a method for the
production of a male transgenic non human mammal, fish or bird
having integrated in its Y or corresponding sex chromosome the
nucleic acid molecule, the regulatory region, the recombinant DNA
molecule or the vector of the invention, a heterologous promoter
controlling expression in spermiogenesis and/or a DNA sequence
encoding an effector (poly)peptide as defined hereinabove alone or
in combination in an active state of expressibility, said method
comprising in vitro fertilization or injection of spermatozoa into
eggs using sperm from said male transgenic non human mammal, fish
or bird. In a preferred embodiment of the present invention, said
method prior to in vitro fertilization or injection further
comprises allowing expression of said effector (poly)peptide and
selecting for sperm expressing said effector (poly)peptide and,
thus, containing said Y or corresponding sex chromosome. The above
method is useful in case the transmission of the construct from
male carriers by natural mating or artificial insemination is close
to 0%. The production of transgenic male carriers can be achieved
by the method of the invention using in vitro fertilization since
it has been shown in mice that transmission ratio distortion of t/+
sperm does not occur during in vitro fertilization. The efficiency
of the method of the invention can be,further enhanced by selection
for sperm carrying a Y or corresponding sex chromosome prior to in
vitro fertilization as described above. Selection can be effected,
e.g., by cell sorting.
[0100] Alternatively, male carriers of the R and/or effector
function which are used for the generation of predominantly female
progeny result from mating of hemizygous male animals carrying an
inactive R and/or effector encoding construct with hemizygous
female animals carrying a locus encoding a site specific
recombinase and preferably the Cre locus. Progeny of such matings
may be used for the maintenance of the strain as well as for the
generation of the desired female progeny. It is worthwhile noting
that from a single male carrier of the R and/or effector encoding
construct many female progeny can be obtained.
[0101] A further embodiment of the invention that has been referred
to above relates to the use of the R gene in combination with
Distorter 2 (D2) preferably in combination with Distorter 1 (D1).
In this embodiment, the chromosome carrying the R construct is
transmitted predominantly or exclusively.
[0102] Distorters D1 and D2 (and possibly D3 as well as further
postulated Distorters) act in trans to the advantage of the
chromosome carrying the R construct. Whereas the applicant does not
wish to be bound by any scientific theory, it is presently assumed
that D1 and D2 are expressed in the diploid phase of
spermatogenesis. Whereas the Distorter genes have not yet been
identified it is well known that their gene products lead to the
predominant or exclusive transmission of the chromosome carrying
the R function. The Distorter function can be provided, for
example, by a chromosome carrying a partial t-haplotype containing,
e.g., Distorter D1 or D2 or both. It is further presumed that the
expression products of the Distorter genes exert a negative
influence on sperm not carrying the R function. In contrast, the
sperm carrying the R function are protected by the R function. It
is also suggested that such sperm may have a selective advantage as
regards motility and thus faster reach the egg cell to be
fertilized.
[0103] It is envisaged in accordance with the present invention
that D2, D1 and further Distorters are located on the same or one
or more different chromosomes than that or those which
carry/carries the R construct. If R is integrated on the Y
chromosome, mating will predominantly result in male progeny.
Integration on the X chromosome, in contrast, will yield
predominantly or exclusively female progeny. Integration in an
autosome will result in a high transmission of said chromosome and
thus any trait linked to said R construct. The high transmission of
the R construct guarantees the maintenance of the R function. A
practical advantage of the embodiment, in the case that the R
encoding construct is integrated in the X chromosome, is that only
few male wild type animals are necessary for the maintenance of the
Y chromosome, i.e., of the male sex. Said male wild type animals
may be generated by mating transgenic hemizygous female animals,
carrying both the Distorter(s) and the R function with wild type
males.
[0104] The subject-matter of the invention relates also to a
transgenic non human mammal, fish or bird having stably integrated
in its genome the nucleic acid molecule, the regulatory region, the
recombinant DNA molecule or the vector of the invention or which is
regenerated from a host cell of the invention or which is
obtainable by the method of the invention referred to above.
[0105] Said transgenic animal is advantageously mouse, cattle,
sheep, pig, goat, rat, rabbit, horse, dog, cat, camel, chicken,
duck, salmon or trout.
[0106] Said transgenic animals may be used for producing offspring
at a non mendelian ratio comprising breeding, in vitro
fertilization or artificial insemination.
[0107] The invention additionally relates to a pair of transgenic
non human mammals, fish or bird, wherein the male is a transgenic
animal having integrated in its Y chromosome the nucleic acid
molecule, the regulatory region, the recombinant DNA molecule, or
the vector of the invention in a reversible inactive state of
expressibility and optionally at least one Distorter in its genome,
and the female is a transgenic animal having stably integrated into
its genomic DNA a nucleic acid molecule encoding a site specific
DNA recombinase.
[0108] The pair of transgenic animals should of course be
preferably of the same species in order to allow a successful
mating.
[0109] Preferably, in said female of said pair of animals, said DNA
recombinase is Cre or flp. Most advantageously, said DNA
recombinase is controlled by regulatory elements that are active
prior to spermiogenesis.
[0110] Further, the present invention relates to sperm obtainable
from a male of the transgenic non-human mammal, fish or bird as
defined herein before.
[0111] Said sperm may be comprised in a composition suitable, for
example, for deep freezing.
[0112] The invention also relates to a method for the selection of
the sperm of the invention comprising allowing expression of the
effector (poly)peptide and selecting for the presence or absence of
said (poly)peptide.
[0113] In accordance with this method of the invention, the
effector (poly)peptide is preferably selected for by cell sorting
or affinity chromatography. Sperm either carrying or not carrying
the effector (poly)peptide and thus the nucleic acid molecule of
the invention may then be used for the further desired purpose.
[0114] Additionally, the invention relates to a method for the
selection against sperm of the invention comprising
[0115] (a) allowing expression of the recombinant DNA molecule
defined herein above that is capable of causing cell death; and
[0116] (b) selecting for viable sperm.
[0117] Cell death can advantageously also be caused by the in vivo
expression of an effector molecule comprising a tag and the
addition of a specific antibody binding to the tag and of
complement to sperm in vitro, resulting in the inactivation or
lysis of the spermatozoa carrying the construct.
[0118] Said methods find applicability in cases where sperm
carrying the R promoter function is to be selected against.
[0119] A further object of the invention is the use of the sperm
for the production of offspring. Such a production may comprise
breeding, in vitro fertilization or artificial insemination.
[0120] An additional object of the present invention relates to the
use of the nucleic acid molecule of the invention, the regulatory
region of the invention, the recombinant DNA of the invention, the
vector of the invention, the host cell of the invention, the
expression product of the invention or the antibody of the
invention for the isolation of receptors on the surface of sperm
recognizing attractants of the egg cell for the development and/or
production of contraceptiva.
[0121] Further, the present invention relates to the use of the
nucleic acid molecule of the invention, the regulatory region of
the invention, the recombinant DNA of the invention, the vector of
the invention, the host cell of the invention, the expression
product of the invention or the antibody of the invention for the
identification of chemicals or biological compounds able to trigger
the (premature) activation or inhibition (repression) of the
signaling cascade in which the Responder function is envisaged to
be involved in. Such compounds could be applicable as potent
contraceptiva since it is envisaged that the activation or
inhibition (repression) of said signaling cascade may affect the
motility of sperm, due to rapid exhaustion of their energy reserve,
and/or by inhibiting sperm movement and/or affect the ability of
sperm to fertilize ovulated eggs.
[0122] The identification of said chemical or biological compounds
could be achieved by standard screening technology using the
activation of the wild type Responder protein expressed in cell
culture cells as an assay. It is e.g. envisaged that activation of
said protein may trigger microtubule disruption in cell culture
cells similar to the effect obtained by overexpression of the MARK
kinase. Compounds triggering or inhibiting such an effect could
then be tested for their effect on the motility and/or
fertilization ability of sperm. Alternatively, a similar screening
system for said compounds could also be envisaged for sperm without
prior employment of a screening assay in cell culture cells.
[0123] Furthermore, the nucleic acid molecule of the invention, the
regulatory region of the invention, the recombinant DNA of the
invention, the vector of the invention, the host cell of the
invention, the expression product of the invention or the antibody
of the invention can be used for the isolation of receptor
molecules and/or other members of the Responder/Distorter signaling
cascade to which said expression product which would be expected to
be a (poly)peptide may bind. Said signal transducing molecules may
be identified by immunoprecipitation of protein complexes involving
the Responder (poly)peptide and cloning of the corresponding genes
encoding them, or by Two Hybrid Screening techniques in yeast
employing standard technology. In particular, most preferably the
Responder gene or (poly)peptide may be used to isolate the membrane
receptor of the signaling molecule which is envisaged to activate
said Responder/Distorter signaling cascade. Said membrane receptor
is envisaged to be most preferable as a target for the development
of novel contraceptives.
[0124] Additionally, the present invention relates to a method for
the detection of the nucleic acid molecule, the regulatory region,
the recombinant DNA molecule, the vector, or the expression product
of the invention or a different heterologous expression product
encoded by said DNA molecule or vector in the transgenic non human
mammal, fish or bird of the invention or a part thereof comprising
identifying said nucleic acid molecule, regulatory region,
recombinant DNA molecule or vector of the invention or a portion
thereof in said transgenic animal or said part thereof. The method
of the invention allows the identification of animals of the
invention on the basis of the genetic constructs they carry in
accordance with the invention. Moreover, the method allows the
identification of such animals e.g. after slaughtering by analyzing
parts thereof. It should be noted that sperm, egg cells and embryos
are also to be considered as parts of said animals. Detection may
be effected by PCR using primers specified herein above. Nucleic
acid hybridization with a detectably labeled probe constitutes a
different method of detection. It is further most important to note
that any portion or component of the nucleic acid, recombinant DNA
molecule or vector may be identified in accordance with the method
of the invention as long as it is indicative thereof. Thus, for
example, the vector may comprise a nucleic acid sequence without
any biological function that is nevertheless indicative of said
vector and thus, of the invention. In another embodiment the
effector (poly)peptide may be used for detection. Of course, the
nucleic acid molecule of the invention or a portion thereof may
itself be detected. All embodiments conceivable by the person
skilled in the art that comprise the above step underfall the
method of the invention as long as they allow the detection of the
above mentioned genetic material.
[0125] Also, the present invention relates to 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.
[0126] Preferred embodiments and various applications of this
method as well as methods of manipulating said sequence or
expression level have been addressed herein before.
[0127] The invention also relates to a transgenic animal having a
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.
[0128] Preferably, said member of said signal cascade is not the
Responder.
[0129] In these embodiments of the invention, the sequence or
expression level of a preferably different member of the cascade
than the Responder is altered or abolished. Simultaneously, it is
expected that the activity of the Responder and/or one or more of
the Distorters is affected. Depending on the type of
alteration/abolishment of Responder/Distorter functions, these
transgenic animals may be used in breeding schemes corresponding to
the ones addressed above.
[0130] Finally, the present invention relates to 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.
[0131] In a preferred embodiment of the invention said genetic
trait determines the sex.
[0132] In another preferred embodiment of the method of the
invention said gene is under the control of a promoter that allows
expression during spermatogenesis/spermiogenesis.
[0133] The promoter may be the original promoter of said gene or
may be derived from a different copy of said gene or from a
different gene. Furthermore, the promoter may be derived from a
copy of the homologous gene (in case more than one exists) from a
different species or may be derived from a different gene from said
different species. The promoters may also be modified in order to
obtain optimum expression, which may be enhanced or reduced
expression.
[0134] In a particularly preferred embodiment of the method of the
invention said promoter allows the preferential or exclusive
expression of said gene in sperm carrying said gene.
[0135] In a further preferred embodiment of the method of the
invention 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.
[0136] In a further preferred embodiment of the method of the
invention said gene encodes an inhibitor of cAMP dependent protein
kinase A.
[0137] In a particularly preferred embodiment said inhibitor is PKI
or a functionally active derivative or fragment thereof.
[0138] As used in accordance with the present invention the term
"functionally active derivative or fragment" denotes molecules that
deviate from PKI by one or more amino acid substitutions,
deletions, and/or additions but essentially retain the biologically
activity/activities of PKI, i.e. retain at least the inhibitory
activity on cAMP dependent protein kinase A. Examples of
functionally active derivatives or fragments of PKI are well known
to the person skilled in the art and can be found, e.g., in
catalogues of biotechnology companies (see, e.g., the Promega
catalogue of 1998).
[0139] In another embodiment, the present invention relates to a
transgenic animal comprising a gene as defined hereinabove.
[0140] Finally, the present invention relates to a sperm obtainable
from the transgenic animal of the present invention.
[0141] The references cited in the present specification are
herewith incorporated by reference.
[0142] The figures show:
[0143] FIG. 1:
[0144] (a) The upper panel shows a schematical drawing of the
extend of the t-chromosome region (thick bars) of complete and
partial t-haplotypes on chromosome 17 of the mouse, as well as the
mapping positions of the Responder (R.sup.t) and two Distorters
(D1, D2) contributing to the transmission ratio distortion
phenomenon (TRD) in mice (Lyon 1984; Fox et al. 1985; Herrmann et
al. 1986; Bullard et al. 1992). The Responder function maps to the
T66B genomic region shown in more detail in the middle panel
(Schimenti et al. 1987; Nadeau et al. 1989; Rosen et al. 1990;
Bullard et al. 1992). The region carrying R is defined by the
recombination breakpoints of the partial t-haplotypes t.sup.h44,
t.sup.h51, t.sup.Jr1 which do not contain R.sup.t, and t.sup.h49 or
t.sup.h2 which do contain R.sup.t. The breakpoints of t.sup.h2 and
t.sup.h49 coincide (Bullard et al. 1992). The intervals within
which the breakpoints must have occurred are not sharply defined
(as indicated by broken lines); only t-haplotype DNA is indicated.
The position of the marker Tu66 serves as an anchor point for
correlating the mapping of the Responder with the genetic fine map
shown on the lower panel. The genomic clones (cosmids cat.15,
ct.184, ct.169, ct.195), restriction map and gene structure of the
fusion of T66Bk and mouse rsk3 demonstrate that the Responder
candidate T66Bk lies well within the region defined as carrying
R.sup.t. The exon-intron structure of T66Bk has not been
determined; black bars indicate restriction fragments containing
exons of mouse rsk3 located in the T66B region (Kispert 1990). The
fragments encoding T66Bk and T66Bk-2 sequences have been determined
by hybridisation of .alpha.-.sup.32P labelled fragment
pCRt.sup.h2-161/170 to cosmid DNA, restriction digested,
electrophoresed and blotted onto Nylon membrane according to
standard techniques and as described in figure legend 2, as well as
by sequencing as described in figure legend 4.
[0145] (b) The analysis of the BamHI fragment B9.1 of cosmid cat.15
demonstrated that another T66Bk gene family member, T66Bk-2, is
located on the centromere-close side of B9.1, whereas the
telomere-close side contains the putative promoter and first exon
of the T66Bk-rsk3 fusion gene. B9.1 contains the complete putative
protein coding region on one exon and a single 3'-exon (indicated
as 3') encoding untranslated sequences of T66Bk-2. The putative
promoter region and first exon encoding untranslated sequences of
T66Bk-2 is located at the centromere-close side of B9.1 probably
within the 6.1 kb BamHI fragment of cat.15, but the exact position
has not been determined.
[0146] Methods:
[0147] The cosmids cat.15, ct.169, ct.184 and ct.195 were isolated
from a cosmid library constructed from t.sup.w12/t.sup.w12 genomic
DNA prepared according to conventional techniques in the vector
pcos2EMBL (Ehrich et al. 1987). Library screening and cosmid
mapping were performed as described (Herrmann et al. 1987; Rackwitz
et al. 1985; Kispert 1990). The restriction map as well as the
structure and sequence of mouse rsk3 have been determined
previously (mouse rsk3 was initially named Tck; Kispert, 1990). The
chromosomal localization of genomic restriction fragments
hybridizing to subfragments derived from cosmids or to cDNA probes
was done by restriction fragment length polymorphism (RFLP) mapping
(Fox et al. 1985; Herrmann et al. 1986). Polymorphic restriction
fragments specific to t-haplotypes were assigned to the T66B region
if present in genomic DNA from t.sup.h2, t.sup.h49, t.sup.low,
t.sup.6, t.sup.w5 or other complete t-haplotypes, but not in DNA
from t.sup.h44, t.sup.h51, or wild type inbred strains, according
to previous characterizations of these t-haplotypes (Lyon 1984; Fox
et al. 1985; Herrmann et al. 1986; Bullard et al. 1992).
[0148] FIG. 2:
[0149] Southern blot hybridization of genomic or cosmid DNA of
various t-haplotype carrying mice, or wild type mouse strains. The
DNA was digested with BamHI endonuclease, blotted on Nylon membrane
and hybridized with the probe pCRt.sup.h2-161/170. Two fragments,
B7.8 and B9.1 (marked by an asterisk), are visualized in
t-haplotypes carrying the Responder, but are absent from
t-haplotypes without R function as well as from wild type strains.
Both fragments are present in the cosmid cat.15 and together
contain the transcription unit of the gene T66Bk, as shown on FIG.
1 (bottom left). B9.1 additionally contains the protein coding and
3'-untranslated region of T66Bk-2. A third hybridizing fragment on
cosmid cat.15 of about 6.1 kb is likely to contain part of the
T66Bk-2 gene. The 6.1 kb BamHI fragment is located at the proximal
(centromere close) end of cosmid cat.15; it is truncated by the
cloning event and thus, it is not identical in size with and cannot
be correlated to any of the fragments identified in the
hybridizations of total genomic DNA.
[0150] Abbreviations: t.sup.Jr1=t.sup.w71Jr1; t.sup.low=t.sup.lowH;
T.sup.Or=deletion chromosome T Oak Ridge 4. 129/Sv, C57BL/6 and
DBA/2 are mouse inbred strains.
[0151] Methods:
[0152] Genomic DNA was prepared as described (Herrmann and
Frischauf, 1987), digested with BamHI, blotted by an alkaline
capillary transfer onto Hybond N+ membrane (Amersham) as described
(Herrmann et al. 1986; Sambrook et al. 1989), UV treated in a UV
Stratalinker 2400 (Stratagene) according to Church and Gilbert
(1984), hybridized in 0.5M NaPi pH 6.8/7%SDS at 68.degree. C. over
night with 2.times.10.sup.6 cpm/ml of probe, washed in 40 mM NaPi
pH 6.8/1%SDS at 68.degree. C., and exposed on Kodak X-AR5 X-ray
film and an intensifying screen at -80.degree. C. The probe was
prepared by random primer extension using the T7 QuickPrime kit
(Pharmacia Biotech), 50 ng of probe DNA and 5 .mu.l of
.alpha.-.sup.32P dCTP (Amersham) at 3000 Ci/mmole according to the
suppliers instructions.
[0153] The cDNA probe fragment pCRt.sup.h2-161/170 was prepared by
standard PCR amplification in 20 mM Tris pH8.4, 50 mM KCl, 1.5 mM
MgCl2, 0.2 mM dATP/dCTP/dGTP/dTTP each, using 1 unit of the Taq DNA
polymerase, approximately 50 ng of the cDNA pCRt.sup.h2-161/144 as
template, 20 pmole of primer 161 and 170 each. 15 cycles of 30
seconds at 94.degree. C., 30 seconds at 50.degree. C. and 2 minutes
at 72.degree. C. with a final extension of 5 min. at 72.degree. C.
were performed, the product was loaded on a 1% agarose gel in TAE
buffer (Sambrook et al. 1989), electrophoresed, the amplified
fragment cut out under long wave length UV light (366 nm) and
purified by centrifugation through an EZ Enzyme Removers column
(Amicon) and ethanol precipitation (Sambrook et al. 1989). The DNA
was dissolved in TE.
[0154] FIG. 3:
[0155] RT-PCR analyses verify that T66Bk maps to the Responder
region and is transcribed during spermiogenesis. a) RT-PCR of
testis RNA with the primer pair 181/144 which is specific for the
T66Bk-rsk3 fusion gene amplifies a cDNA fragment of 821 bp from RNA
of t-haplotypes carrying the t-Responder (for comparison see FIG.
1) confirming that this gene is present in the t-Responder carrying
region and is expressed in testis (upper panel). The quality of the
RNA and first strand cDNA used for the assay was confirmed by
RT-PCR with the primer pair 145/146 which amplifies a cDNA fragment
of 769 bp from the mouse rsk3 gene (Tck, see Kispert 1990). The
latter RT-PCR also produces a smaller fragment in t-haplotypes
containing the T66B region, but not in wild type or t-haplotypes
which do not contain the T66B region. This smaller cDNA fragment is
due to the deletion of an exon in the T66B-copy of rsk3. A
substantial level of transcription of the T66Bk-rsk3 fusion gene is
first detectable in 22 days p.p. testis (lower panel). At this
stage haploid spermatids have formed and are undergoing the
transformation process into spermatozoa called spermiogenesis (Rugh
1990). The primer pair 155/170 amplifies a cDNA fragment of 815 bp
derived from T66Bk as well as related genes. The presence of RNA at
all stages of spermatogenesis tested with the primer pair 155/170
suggests an early onset of the transcription of one or several
members of the T66Bk gene family. A very low (basal) level of
transcript from the T66Bk-rsk3 fusion gene is also detectable in
early stages of spermatogenesis. b) Comparative RT-PCR of testis
RNA with primer pairs specific for testis specific transcripts of
angiotensin converting enzyme (ACE, Howard et al. 1990), c-kit
(Rossi et al. 1992) and mouse protamine 1 (mP1, Peschon et al.
1987) allows a correlation of the transcription of the T66Bk-rsk3
fusion gene with that of known genes. The promoters of all three
genes have been analyzed in transgenic mice (Langford et al. 1991;
Albanesi et al. 1996; Peschon et al. 1987). mP1 is supposed to be
transcribed in round, ACE and c-kit in elongating spermatids.
Since, in our RT-PCR analysis the T66Bk-rsk3 fusion gene appears to
be transcribed slightly later than ACE and c-kit we conclude that
expression of the T66Bk-rsk3 fusion gene most likely commences in
elongating spermatids.
[0156] Methods:
[0157] Total RNA of testis tissue was prepared following
homogenization of the tissue in LiCl/urea according to a published
procedure (Auffray and Rougeon 1979). After ethanol precipitation
the RNA was dissolved in 50 .mu.l 10 mM Tris-HCl/1 mM EDTA pH7.6
(TE) per approximately 100 mg starting material. 2 .mu.l total RNA
(appr. 6 .mu.g RNA) were used for cDNA synthesis with an oligo(dT)
primer according to the instructions of the SuperScript plasmid
cDNA synthesis kit of Gibco/BRL. After first strand synthesis the
reaction was diluted to 50 .mu.l with TE. For PCR amplification 0.5
.mu.l of the first strand cDNA stock solution was added to 20 .mu.l
of the reaction mix containing 20 pmole of each primer, 20 mM Tris
pH8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dATP/dCTP/dGTP/dTTP each,
and 1 unit Taq DNA polymerase. Reaction mixes were overlayed with
mineral oil and 35 cycles of 30 seconds at 94.degree. C., 30
seconds at 50.degree. C. and 30 seconds at 72.degree. C. were
performed using a PTC-100 thermocycler (MJ Research, Inc.). The
reaction products were electrophoresed in 1% or 2% agarose gels, as
applicable, containing 0.4 .mu.g/ml ethidium bromide in TAE buffer
(Sambrook et al. 1989), and photographed on a UV light box. The 1
kb ladder of Gibco/BRL was used as marker, as shown on the left
margin of each photograph.
[0158] FIG. 4:
[0159] a) Nucleic acid and amino acid sequence of
pCRt.sup.h2-161/144, representing a partial cDNA of the T66Bk-rsk3
fusion gene encoding a putative protein of 484 amino acid residues.
Several in frame stop codons 5' to the first methionine (start
codon) and the stop codon at the end of the single long open
reading frame suggest that the protein coding region of this cDNA
is complete. However, the 5' and 3' non-coding sequences are most
likely incomplete. An asterisk indicates the junction between the
T66Bk gene and the truncated mouse rsk3 gene. Nucleic acid
sequences of primers used for RT-PCR detection and cloning of T66Bk
sequences are indicated. The primer number and 3' end are
given.
[0160] b) Partial nucleic acid sequence of a cDNA fragment,
ptlib0.7, consisting of a fragment from the 5' end of a
T66Bk-related gene fused to part of a mouse rsk3-related gene. This
partial cDNA was isolated by PCR amplification with a plasmid
vector anchor primer (seq5lib) and primer 144, from clone pools of
a total of approximately 200,000 clones of a cDNA plasmid library
constructed with RNA extracted from testis of a t.sup.w5/t.sup.w12
adult male. Another 380,000 cDNA clones were screened by cDNA
filter hybridization. From those clones another partial cDNA
containing a sequence homologous to the one shown here, fused to
rsk3 sequences, was obtained. A primer (161) located at the 5' end
of the cDNA sequence shown was designed and used in combination
with primer 144 (rsk3) to amplify the cDNA fragment of T66Bk shown
on FIG. 4a, from testis cDNA of a t.sup.h2/t.sup.h2 adult
mouse.
[0161] Methods:
[0162] A cDNA library of testis RNA of an adult male carrying the
complete t-haplotypes t.sup.w5/t.sup.w12 was constructed in the
plasmid vector pSV-Sport1 using the SuperScript Plasmid cDNA
synthesis kit (Gibco/BRL) according to the suppliers instructions.
RNA isolation was performed as described in the legend to FIG. 3,
mRNA purification was done using Oligotex beads according to the
supplier's instructions (Qiagen). DNA preparations of library pools
of a total of appr. 200,000 clones were prepared with the Qiagen
plasmid midi kit (Qiagen) and tested by PCR amplification as
described in figure legend 3 using primer pair seq5lib/144. A
fragment of 0.7 kb was obtained and cloned in the vector pCR2.1
using the TA cloning kit of Invitrogen according to the instruction
manual. Another 380,000 cDNA clones were plated on filters and
screened by hybridization as described (Herrmann et al. 1987).
[0163] The partial cDNA pCRt.sup.h2-161/144 was obtained by PCR
amplification of cDNA, prepared and amplified as described in
figure legend 3, except that the primer extension time at
72.degree. C. was 2 minutes per cycle, from testis RNA of an adult
male homozygous for the t-haplotype t.sup.h2, with the primer pair
161/144. The cDNA fragment was purified from a 1% agarose gel as
described in figure legend 3, and cloned in the plasmid vector
pCR2.1.
[0164] Plasmid DNA was prepared with the Qiagen Plasmid Midi kit.
Sequencing reactions were performed using the RR DyeDeoxy
Terminator Cycle Sequencing kit (PE Applied Biosystems) according
to the instructions and gene specific primers (MWG Biotech)
designed with the OLIGO Primer Analysis Software (NBI), the
reactions were purified by centrifugation through Centri-Sep
columns (Princeton Separations) according to the instructions, and
run on an automatic ABI Prism 310 Genetic Analyzer (PE Applied
Biosystems). Sequences were evaluated with the MacMolly Tetra
programs set (Soft Gene, Berlin) on a Power Macintosh computer.
[0165] FIG. 5:
[0166] Northern blot hybridization demonstrating the transcription
of T66Bk-gene family members. Transcripts are detectable in adult
testis from all t-haplotype or wild type strains tested, but not in
RNA from any other organ tested. During spermatogenesis a
detectable level of transcript first appears at 22 days p.p. For a
control the blot was re-hybridized with a probe for GAPDH (Kispert
1990).
[0167] Methods:
[0168] RNA was extracted as described (Auffray and Rougeon 1979),
10 .mu.g per lane was loaded on a 1% agarose gel containing
formaldehyde and electrophoresed in MOPS buffer according to
standard techniques (Sambrook et al. 1989). The gel was washed
twice for 20 minutes in 0.1M NH4-acetate, once in 50 mM NaPi buffer
pH 6.8, in 2 gel volumes each, and blotted onto Hybond N+ membrane
(Amersham) by capillary transfer (Sambrook et al. 1989) using a
reservoir of 50 mM NaPi buffer pH 6.8. The filter was UV treated in
a UV Stratalinker 2400 (Stratagene) according to Church and Gilbert
(1984), hybridized with 2.times.10.sup.6 cpm/ml of the probe
pCRt.sup.h2-161/170 in 0.5M NaPi buffer pH 6.8/7%SDS/25% formamide
at 68.degree. C. over night, washed in 50 mM NaPi buffer pH
6.8/1%SDS at 68.degree. C. and exposed on Kodak X-AR5 film using an
intensifying screen. The probe fragment was amplified by PCR with
the primer pair 161/170 using the cDNA pCRt.sup.h2-161/144 as
template and labeled as described in figure legend 2. To determine
the relative amount of RNA in each lane the filter was
re-hybridized as above with the cDNA clone pme66 containing a
partial cDNA of the GAPDH gene (Kispert 1990).
[0169] FIG. 6:
[0170] Southern blot hybridization of DNA derived from several
mammalian species and the chick, with the probe pCRt.sup.h2-161/144
demonstrates the presence of T66Bk-related genes in hamster,
rabbit, pig, human and chick suggesting the conservation of this
gene class during evolution. The DNA was digested with BamHI,
blotted on Nylon filter, and hybridized and washed at reduced
stringency (58.degree. C.).
[0171] Methods:
[0172] Genomic DNA was isolated from organs or blood cells (human)
as described (Herrmann and Frischauf 1987), cut with BamHI
endonuclease, electrophoresed in a 1% agarose gel in TBE buffer and
blotted by alkaline capillary transfer as described (Sambrook et
al. 1989; Herrmann et al. 1986) onto a Hybond N+ membrane
(Amersham). The filter was UV treated in a UV Stratalinker 2400
(Stratagene) according to Church and Gilbert (1984), hybridized
with 2.times.10.sup.6 cpm/ml of the probe pCRt.sup.h2-191/144 in
0.5M NaPi buffer pH 6.8/7%SDS at 58.degree. C. over night, washed
in 100 mM NaPi buffer pH 6.8/1%SDS at 58.degree. C. and exposed on
Kodak X-AR5 film using an intensifying screen. The probe fragment
pCRt.sup.h2-161/144 was labeled as described in figure legend
2.
[0173] FIG. 7:
[0174] The mouse genome contains several members of the T66Bk gene
family.
[0175] a) The protein coding exon of one member, T66Bk-2, is
located in a tandem duplication arrangement on the centromere-close
side of T66Bk, contained in the BamHI fragment B9.1 of the T66B
region cosmid cat.15. The nucleotide and putative amino acid
sequence of this exon are shown (FIG. 7a). The sequence of primer
232 and 237 used for cDNA detection, mapping and expression studies
(see FIG. 8) are indicated by a dashed line. A single base which is
deleted in the cDNA T66k-8 (T 1164) is underlined.
[0176] b) The cDNA T66k-8 was isolated from a testis cDNA library
of the genotype t.sup.w5/t.sup.w12. Its nucleotide sequence is
identical to that of T66Bk-2 in the region of overlap except for a
single base deletion resulting in a shift of the open reading frame
from amino acid residue 359 onwards (underlined). The sequences for
primer 161 and 237 are indicated (see FIG. 8).
[0177] c) The cDNA T66k-7as is derived from an antisense transcript
of a T66Bk family member. The 5' end of T66k-7as is closely related
to sequences upstream of the T66Bk promoter. Its 3' end is very
similar to the 5'intron near the protein coding exon of
T66Bk/T66Bk-2 (see FIG. 7a). The location of T66k-7as in the genome
has not been determined. Vector sequences are underlined by a
dashed line, sequences with a high similarity to the exon encoding
the large ORF of T66Bk/T66Bk-2 by a double dashed line, sequences
with high similarity to intron sequences upstream or downstream of
the protein coding and 3'-untranslated exon, respectively, of
T66Bk/T66Bk-2 by """. The direction of transcription of the
T66Bk/T66Bk-2 homology region is indicated.
[0178] d) The cDNA clone, T66k-20, was isolated from the
t.sup.w5/t.sup.w12 testis cDNA library. The nucleotide and putative
amino acid sequence shows a strong similarity to the above members
of the T66Bk gene family.
[0179] e) Comparison of the putative amino acid sequences of the
members of the T66Bk gene family. Amino acid residues identical to
T66Bk are indicated by ". Gaps indicated by _ were introduced to
allow optimal alignment. Note the strong similarity of all protein
sequences as well as the altered protein tail in T66k-8. Note also
the closer relationship of T66Bk-2 and T66k-20 compared to T66Bk,
despite the fact that T66k-20 is longer at the N-terminus.
[0180] Methods:
[0181] The BamHI fragment B9.1 of cosmid cat.15 was isolated by
restriction digestion and cloned in the vector pBluescript KS
according to standard techniques. The DNA preparation and
sequencing was carried out as described in Figure legend 4. The
cDNA clones T66k-7as, T66k-8 and T66k-20 were isolated from a cDNA
library constructed from testis of a t.sup.w5/t.sup.w12 male, the
library plated and screened by hybridization with a cDNA fragment
derived from PCR amplification of the cDNA pCRt.sup.h2-161/144 with
the primer pair 155/170. Library screening, probe preparation,
hybridization, plasmid preparation, sequencing etc. are described
in figure legends 2, 3 and 4.
[0182] FIG. 8:
[0183] The T66Bk-2 gene is located in the T66B region and is
expressed from 22 day p.p. in the testis.
[0184] A cDNA fragment of 951 bp derived by RT-PCR amplification of
testis RNA and hybridization with a T66Bk-2/T66k-8 specific primer
(232) is detectable in RNA derived from mice carrying the
t-haplotypes t.sup.h2, t.sup.h49, t.sup.6 and t.sup.w5 but not in
t.sup.h44, t.sup.h51 and t.sup.Jr1. Therefore it maps to the T66B
region, in agreement with the mapping data of cosmid cat.15. The
signal obtained from t.sup.h2/t.sup.h2 and t.sup.h49/t.sup.h49 is
higher than that obtained from T.sup.Or/t.sup.6 or
T.sup.Or/t.sup.w5 in agreement with the fact the former two are
homozygous for T66Bk-2, while the latter are heterozygous. A faint
signal is obtained in t-haplotypes carrying the T66A region only or
in wild type (Balb/c). This is due to a reduced capability of
binding of the oligonucleotide 232 to other members of the T66Bk
gene family. In testis RNA derived from t.sup.6/+ males of
different stages (lower panel) T66Bk-2 transcription is first
detected at 22 days p.p. However, the signal is very weak, but is
significantly increased at 24 days p.p. This suggests that T66Bk-2
may be expressed at a lower level or later than T66Bk. Overall, the
transcription level of T66Bk-2 in each testis sample detected by
RT-PCR and hybridization correlates well with the number of T66Bk-2
alleles present in each of the samples. This together with the
sequence conservation further suggests that the cDNA clone T66k-8
is derived from the locus T66Bk-2 within the T66B region.
[0185] Methods:
[0186] RNA derived from testis was reverse transcribed, first
strand cDNA was amplified by PCR using the primer pair 161/237 (see
FIGS. 7a, b), and the products separated by electrophoresis on 1%
agarose as described in figure legend 3. The cDNA was transferred
to Hybond N+ filters as described in figure legend 2, and
hybridized with oligonucleotide 232 labeled using the DIG
Oligonucleotide Tailing Kit (Boehringer Mannheim) according to the
instructions of the supplier. Hybridization was carried out in 0.5M
NaPi pH 6.8/7% SDS at 37.degree. C. The filters were washed 4 times
for 5 minutes in prewarmed 40 mM NaPi pH 6.8/1% SDS (37.degree. C.)
at room temperature. Prehybridization and oligonucleotide detection
were done according to the protocol from Boehringer (Mannheim).
[0187] FIG. 9:
[0188] Nucleic acid and amino acid sequence of a cDNA encoding the
T66Bk gene.
[0189] The sequence extends the sequence of pCRt.sup.h2-161/144
shown on FIG. 4a, both at the 5'- and at the 3'-side, but is
identical in the region of overlap. The 3'-end of the cDNA
pSV-T66Bk ends in an intron of the mouse rsk3 gene and lacks a
consensus polyadenylation signal suggesting that it was derived by
oligo(dT) priming of incompletely spliced RNA. Asterisks indicate
the positions of introns. The asterisk between position 2023 and
2024 indicates the fusion point between MARK- and rsk3-homology
regions of T66Bk.
[0190] Methods:
[0191] Another cDNA library, in addition to the one used to isolate
cDNAs presented on FIG. 7, was constructed from testis RNA of a
male carrying the complete t-haplotypes t.sup.6/t.sup.w5 according
to the methods described in figure legend 4 and screened as
described in figure legends 7, 2, 3 and 4. A total of 500 000 cDNA
clones contained in 10 pools were analysed by PCR for the presence
of cDNA clones encoding the gene T66Bk using the primer pair
161/144. Four positive clones were identified and one, named
pSV-T66Bk, was purified by colony hybridization screening using the
cDNA pCRt.sup.h2-161/144 as probe, and sequenced.
[0192] FIG. 10:
[0193] Nucleic acid and putative amino acid sequences of wild type
members of the T66Bk kinase gene family.
[0194] a) The cDNA pCR.Balb-66k was isolated by RT-PCR from testis
RNA of the wild type inbred mouse strain Balb/c. The putative start
codon of the open reading frame is located 20 amino acid residues
further upstream from the translation start of the T66Bk gene, very
similar to the situation observed in T66k-20. The ORF is equal in
length to that of T66k-20. Since in both genes, pCR.Balb-66k and
T66k-20, the putative translation start does not conform closely
with Kozak's rules it is possible that this start codon of
translation is not efficiently used. Thus, it might be that either
this or the next 3'-located translation start codon or both are
utilized.
[0195] b) The cDNA pCR.C3H-66k was isolated by RT-PCR from testis
RNA of the wild type inbred mouse strain C3H/N using the primers
161/220. In contrast to the ORF of T66Bk, the ORF of this gene is
shorter at the C-terminal end resulting in a putative protein of
433 amino acid residues.
[0196] c) This is also the case for the ORF encoded by the genomic
clone fragment p.lambda.. 129-66k derived from the 129Sv wild type
inbred mouse genome. The significance of this alteration of the ORF
compared to the gene T66Bk is unclear. However, it is assumed that
the length of the ORF and thus the resulting protein sequence may
influence the properties of the protein.
[0197] FIG. 11:
[0198] Nucleic acid sequence of the putative promoter of the gene
T66Bk. The BamHI fragment B9.1 of the cosmid cat.15 contains the
protein coding region of T66Bk-2 (see FIGS. 2 and 7) as well as the
putative transcription start site and upstream region of T66Bk. The
sequence of 3641 bp presented here shows the intron and
3'-untranslated exon of T66Bk-2, located 3' of the T66Bk-2 sequence
shown on FIG. 7, followed by the upstream region and putative first
exon of T66Bk. Splice donor/acceptor sites are indicated by an
asterisk (*). Exon sequences are underlined. The underlined exon
sequence of T66Bk shown represents the sequence contained in the
cDNA pSV-T66Bk; the transcription start site of T66Bk, however, may
be located further upstream. Two consensus TATA boxes are shown in
bold type and underlined. The transcription start site of T66Bk has
not been determined, but is likely to be located 3' of either of
the TATA boxes. It cannot be excluded that both TATA boxes are
utilized alternatively for binding of the TATA binding protein
complex. The restriction sites for KpnI and PmII used to isolate
the putative promoter fragment utilized in the construction of tg5
are indicated in bold type. The sequence contains a number of
potential binding sites for known transcription factors (Faisst and
Meyer 1992). However, since none of them have been demonstrated to
be functional, they have been omitted on the figure. Their
positions can be readily identified by sequence analysis software
such as MacMolly's Interpret program (Soft-gene, Berlin).
Regulatory elements conferring tissue and stage specific regulation
of transcription are often located just upstream of the
transcription initiation sites, but may also be located in the
first exon, intron or at a distance either far upstream or
downstream. It is not known whether the sequence shown here
contains all cis-regulatory elements or only a subset required for
specific expression of T66Bk during spermiogenesis. It is also
envisaged that the long 5'-untranslated region of T66Bk mostly
comprised by exon 1 may have a function in regulating the onset
and/or efficiency of translation.
[0199] Methods:
[0200] Cloning and sequencing of BamHI fragment B9.1 were done as
described in figure legend 7.
[0201] FIG. 12:
[0202] The transgenes tg4 and tg5 are expressed during
spermiogenesis.
[0203] To confirm that the transgenes tg4 and tg5 which showed
distortion of their transmission from male carriers to their
offspring are expressed in the testis, RT-PCR analysis was carried
out using a transgene specific primer pair. For tg4 the primer pair
309/310 amplifying a junction fragment between the MARK- and rsk3
homology regions was used. For tg5, the primer pair 313/314
amplifies its 3'-end from hCD24 to the polyadenylation signal
sequence. Various post partum stages of testis expected to be in
the process of spermatid maturation were analyzed. mRNA was DNAsel
treated before reverse transcription and 1 .mu.l of this solution
was amplified by PCR (+DNAsel/-RT). After reverse transcription of
the remainder, 1 .mu.l of it was amplified in parallel. Tg5-43 was
tested with 313/314 except for tg5-43 stage 39 days p.p. which was
control tested with the primer pair 309/310.
[0204] None of the control reactions showed a PCR product, whereas
all samples subjected to reverse transcription yielded the expected
fragment after PCR. This demonstrates the expression of tg4 and
tg5, respectively, in the testes of male carriers. However,
expression occurs earlier than expected from the analysis of c-kit
and T66Bk shown on FIG. 3. This might be due to the sensitivity of
the RT-PCR assay which might detect basal transcription of the
transgenes, or to inappropriate control of transgene expression
caused by the promoter fragment used in the construction or caused
by influences of the integration sites. On the other hand, the
adult male carrying tg4-3 and the tg5-43 39 day p.p. male showed a
stronger fragment suggesting an increase of transgene expression
during maturation or following mating to females. Abbreviations:
ad, adult male (mated); M, marker (1 kb ladder (Gibco/BRL)
[0205] Methods:
[0206] RT-PCR was carried out essentially as described in figure
legend 3 with the following exceptions. Before addition of Reverse
Transcriptase to the reaction 1 .mu.l of DNAsel (RNAse free, 10
units/.mu.l) was added and the reaction was incubated at 37.degree.
C. for 20 min. 1 .mu.l of the reaction was removed and kept on ice,
to the remainder 1 .mu.l of Superscriptil Reverse Transcriptase
(200 units/.mu.l, Gibco/BRL) was added and the reaction was
incubated for a further 20 min. each at 37.degree. C. and
55.degree. C. All PCR reactions were set up with the same PCR stock
solution to which 1 .mu.l of either the control reaction
(+DNAsel/-RT) or the test reaction (+DNAsel/+RT) were added. PCR
using the primer pair 309/310 was carried out as described in table
1 legend. The same conditions were used for the primer pair 313:
5'-ATGGGCAGAGCAATGGT-3' and 314: 5'-CAGGTTCAGGGGGAGGT-3'.
[0207] FIG. 13:
[0208] T66Bk contains a second ORF encoding an N-terminal
polypeptide of mouse rsk3.
[0209] The figure shows the cDNA sequence of pSV-T66Bk emphasizing
the ORF encoded by the rsk3 homology region. The putative
translation start and stop codons of the MARK-homology region as
well as two potential translation start codons of the rsk3 homology
region are underlined. The amino acid sequence shown starts at an
ATG codon located 3'of the stop codon of the MARK related kinase
and 5'of the splice site, indicated by an *. Another potential
translation start codon is located in the rsk3 homology region.
Although unlikely, there are two possibilities that this ORF is
translated. First, the ribosome might not fall off the mRNA after
completing translation of the MARK-related kinase and re-start
translation at the next ORF. Second, alternative splicing might
skip the exon encoding the MARK-related kinase. This would result
in a transcript in which the ATG at position 2107-2109 would be the
first potential translation start site. The latter is the case
observed in the partial cDNA sequence ptlib0.7 shown on FIG. 4b
demonstrating that such transcripts exist. However, they are not
observed in males carrying the t-haplotypes t.sup.h2 or t.sup.h49,
but only in complete t-haplotypes sugging that they are derived
from a gene located outside of the region carrying the
t-Responder.
[0210] The examples illustrate the invention.
EXAMPLE 1
Cloning of a Novel Candidate Gene for the T Complex Responder
[0211] Cosmid clones from the T66B region were isolated and their
genomic location within T66B verified by RFLP mapping (FIG. 1). In
particular, the fragment pAK34 which is contained within the
overlap of the cosmids ct.184 and cat.15 hybridizes to 3 genomic
BamHI fragments in complete t-haplotypes, of which one, a 5.5 kb
fragment, is located in the T66B region (Kispert 1990). The cosmids
ct.184 and cat.15 contain the 5.5 kb BamHI fragment hybridizing to
probe pAK34, thus confirming that they are derived from the T66B
region. Likewise, the PCR fragment 161/170 derived from the cDNA
described here hybridizes to the BamHI fragments B9.1 and B7.8
contained within cosmid cat.15, and both can be mapped to the T66B
region (FIGS. 1 and 2).
[0212] A gene spanning at least 60 kb of the genomic region
contained within the cosmid cluster isolated from the T66B region
was identified. This gene is represented in 3 copies in
t-haplotypes, one each in the regions T66A, T66B and T66C. The wild
type form of it encodes the mouse homologue of human rsk3 (Zhao et
al. 1995), a kinase of the pp90 ribosome S6 kinase family (called
Tck in Kispert 1990). The gene copy located in the T66B region is
altered compared to wild type (Kispert 1990). The 5' end is not
contained within cosmid cat.15 and one additional exon is missing.
The fact that one additional exon is missing was detected by RT-PCR
of testis RNA derived from a panel of partial and complete
t-haplotypes and wild type with the primer pair 145/146. In
addition to the expected fragment of 769 bp a smaller fragment was
obtained in the t-haplotypes containing the T66B region, but not in
those containing only T66A nor in wild type. (FIG. 3a). This
demonstrated that the T66B gene copy of rsk3 is expressed in
testis. To identify the 5' sequence of this gene, a cDNA library
was constructed from mRNA of the testis of a t.sup.w5/t.sup.w12
male mouse. Surprisingly, two clones were isolated from a total of
approximately 580000 cDNA clones screened which contain
heterologous sequences 5' to base 438 of wild type rsk3 (Kispert
1990). The partial sequence of one of these clones is shown on FIG.
4b. Primers for polymerase chain reaction (PCR) amplification were
designed such that the forward primer (161) is located at the 5'
end of this cDNA, that is within the novel sequence, and the
reverse primer (144) is located in the rsk3 sequence. PCR
amplification of testis cDNA prepared from RNA of the partial
t-haplotypes t.sup.h2 and t.sup.h49 produced a fragment of 2.1 kb,
whereas no band was detected in t.sup.h44, t.sup.h51, t.sup.Jr1 or
BALB/c (wild type) cDNA. The fragment (pCRt.sup.h2-161/144) was
isolated from t.sup.h2, cloned and sequenced (FIG. 4a). It
comprises yet another novel gene located within the T66B region
(see below).
[0213] A primer pair (181/144) designed on the basis of the
sequence of pCRt.sup.h2-161/144 allows the amplification of a cDNA
fragment of a testis expressed gene which is contained in t.sup.h2,
t.sup.h49, t.sup.w5 and t.sup.6, but not t.sup.h44, t.sup.h51,
t.sup.Jr1 or BALB/c (wild type) testis (FIG. 3a). Thus the
corresponding transcript is t-specific and derived from a gene
mapping to the T66B region. RT-PCR with the primer pair 145/146 for
mouse rsk3 also confirmed the quality of the first strand cDNA
synthesis. The cDNA-mapping by PCR confirms the genomic
localization by Southern blot hybridization (see FIGS. 1 and
2).
EXAMPLE 2
The t Complex Responder Candidate Gene Encodes a Novel Kinase
[0214] The sequence of the 2.1 kb cDNA fragment pCRt.sup.h2-161/144
contains a single long open reading frame (ORF) encoding a protein
of 484 amino acid residues (FIG. 4a). Several "in frame" stop
codons upstream of the first potential translation start codon
(bases 337-339) suggest that the N-terminal end of the putative
protein is complete. The translation stop (bases 1789-1791) is
still located within the "non-rsk3" sequence; the rsk3 sequence of
the fusion transcript starts at base 1837.
[0215] Sequence comparisons with protein sequence databases
revealed several known motifs within the ORF, most importantly a
protein kinase domain and a consensus protein tyrosine kinase
active site. However, the pattern of conserved residues is more
strongly related to the consensus for serine/threonine kinases,
suggesting that the isolated gene encodes a novel Serine/threonine
kinase. However, the in vivo specificity remains to be determined
experimentally. In accordance with the present invention, the gene
is called T66Bk. The best match to known kinases was found to MARK,
a recently published serine/threonine kinase which is involved in
the regulation of the cytoskeleton (Drewes et al. 1997). The
identity to MARK2 is more than 25% and approximately 38% at the
amino acid level within the putative kinase domain. The putative
protein contains 8 potential phosphorylation sites for casein
kinase II, 5 for protein kinase C and 5 potential myristoylation
sites.
[0216] The data explained above suggest that the T66Bk-rsk3 fusion
gene arose by a rearrangement event resulting in the fusion of two
gene parts, both derived from a kinase. The 5' region probably
including the transcriptional control elements are derived from a
MARK related kinase. The 3' end which is derived from the mouse
rsk3 gene and may include most of its sequence and probably also
its poly(A) addition signal might be around 5 kb long. The Southern
blot hybridization data shown in FIG. 2 suggest that the genome may
contain several gene family members of the MARK-related kinase.
EXAMPLE 3
Transcripts Derived from T66Bk-Gene Family Members Accumulate
During Spermiogenesis
[0217] In a Northern blot hybridization assay transcripts derived
from T66Bk related genes can be detected in 22 day post partum
(p.p.) male t.sup.6/+ testis or later, using the cDNA fragment
pCRt.sup.h2-161/170 as a probe (FIG. 5). Two transcripts of
approximately 2.8 kb and 3.2 kb can be distinguished in
T.sup.Or/t.sup.6 and T.sup.Or/t.sup.w5 testis RNA. Only the lower
band is clearly detectable in BALB/c (wild type) testis RNA. This
difference may be caused by differential splicing or different
sequence of gene variants which distinguish, for example,
t-haplotypes and wild type or various wild type strains. As the
expected transcript size of the T66Bk-rsk3 fusion gene is appr. 7
kb, an assignment of one of the observed RNA bands to the
T66Bk-rsk3 fusion gene is not possible. The Northern analysis
showed that the members of the T66Bk gene family are fairly
specifically expressed, and might even be restricted to the testis,
as no transcripts were detected in RNA isolated from ovary, liver,
spleen, kidney, lung or heart.
[0218] In a RT-PCR analysis of testis RNA using the primer pair
155/170, transcripts are detectable as early as day 7 p.p., the
earliest stage of spermatogenesis tested (FIG. 3a). This suggests
that low level transcription of one or several T66Bk-related kinase
genes occurs early during spermatogenesis, but high level
transcription detectable by Northern analysis occurs during
spermiogenesis.
[0219] In agreement with this interpretation, very low (basal)
levels of transcripts of the T66Bk-rsk3 fusion gene are detectable
by RT-PCR at stage 7, 14 and 20 days p.p., but much higher levels
can be seen only from stage 22 d.p.p. onwards (FIG. 3a). This
suggests that the T66Bk-rsk3 fusion gene is up-regulated at about
the stage when elongating spermatids appear (see below).
[0220] The genes mouse protamine 1 (mP1), angiotensin converting
enzyme (ACE) and c-kit were analyzed in order to allow a staging of
the onset of the T66Bk-rsk3 fusion gene expression during
spermatogenesis (FIG. 3b). mP1 has been reported to be first
expressed in round spermatids (Peschon et al. 1987), the testis
specific promoters of ACE (Howard et al. 1990) and c-kit (Rossi et
al. 1992) are first active in elongating spermatids of undefined
stage and stage IX-XI, respectively. The analysis of all three
promoters has been achieved using transgenic animals (Langford et
al. 1991; Albanesi et al. 1996; Peschon et al. 1987). In the RT-PCR
analysis shown here, mP1 transcripts were detected as early as day
14 p.p., but a strong band appeared at day 18 p.p. According to
Rugh (1990), spermatids appear at day 17 p.p. in male pups. The ACE
and c-kit testis transcripts were weakly detectable at 20 days
p.p., but a signal comparable to the T66Bk-rsk3 fusion gene band
181/144 first appeared at 22 days p.p. An earlier expression of ACE
was detected in day 7 and 14 p.p. testis. Thus, the RT-PCR data are
in agreement with the published data showing that ACE and c-kit are
expressed in elongating spermatids. This suggests that the
expression of the T66Bk-rsk3 fusion gene in testis is up-regulated
at about the same time or a little later than that of c-kit and
ACE, in elongating spermatids, and that the promoter of the
T66Bk-rsk3 fusion gene may be active late enough during
spermiogenesis to exclude the distribution of the T66Bk-rsk3 fusion
gene products to spermatocytes not containing the T66Bk-rsk3 fusion
gene (Willison and Ashworth 1987), thus fulfilling an important
criterion for the R function. The low level of expression found in
day 7 and 14 p.p., but not in day 18 p.p. testis suggests that the
transcripts might be degraded by the end of meiosis.
EXAMPLE 4
T66B-Related Genes Are Conserved During Evolution
[0221] Putative homologs of the T66Bk-related kinases also exist in
other species (FIG. 6). A Southern blot hybridization assay at
reduced stringency using the cDNA fragment 191/144 as a probe
revealed cross-hybridizing fragments in hamster, rabbit, pig, chick
and human. This suggests a conservation of the T66Bk-related
kinases in other mammals as well as in birds.
EXAMPLE 5
The Mouse T/t-Complex Encodes Several Members of the T66Bk Gene
Family
[0222] In a Southern blot hybridization of cosmid cat.15 with the
probe pCRt.sup.h2-161/170 three hybridizing BamHI fragments, B7.8,
B9.1 and a 6.1 kb BamHI fragment are detected (see FIG. 2).
Sequencing of the T66Bk or related gene encoding parts of these
genomic DNA fragments revealed that each of the BamHI fragments
B7.8 and B9.1 contains a large open reading frame (ORF) encoding
T66Bk and another member of the T66Bk gene family, respectively.
The centromere farthest BamHI fragment (B7.8) contains the T66Bk
ORF (FIGS. 1 and 4a). Its transcribed part (exon) differs from the
corresponding exon contained in the cDNA pCRt.sup.h2-161/144 by a
single point mutation (base 1490 C to T) probably due to an allelic
variation between the t-haplotypes t.sup.h2, and t.sup.w12 from
which cosmid cat.15 was derived, resulting in a single amino acid
exchange (Pro to Leu).
[0223] The next centromere closer BamHI fragment (B9.1) contains
5'-noncoding sequence and most likely the promoter of T66Bk and,
further upstream of it, an ORF encoding exon and a 3'-noncoding
exon of another member of the T66Bk gene family, named here
T66Bk-2. However, in this case the 3'-noncoding exon is not related
to rsk3. The exon sequence of T66Bk-2 encoding a large ORF is shown
on FIG. 7a. It differs from the ORF of T66Bk in a number of
positions; nevertheless, it is very closely related to T66Bk. In
the t.sup.6/+ testis cDNA panel, expression of T66Bk-2 is first
detected at 22 days p.p. Considerably higher expression is observed
from 24 days p.p. onwards (FIG. 8).
[0224] The mouse genome contains several more loci of the T66Bk
gene family some of which are located in the region of the
T/t-complex distal to T66B, probably in T66C. This is based on the
observation of several BamHI fragments hybridizing to
pCRt.sup.h2-161/170, other than those described above, contained in
the genome of mice carrying partial t-haplotypes or wild type mice.
Some of these BamHI fragments are polymorphic and specific to
complete t-haplotypes, but are not present in the partial
t-haplotypes t.sup.h44, t.sup.Jr1, t.sup.lowH, t.sup.h2 or
t.sup.h49 nor in wild type (see FIG. 2). Therefore they must be
contained in the T/t-complex region distal to T66B. To obtain
coding sequences of T66Bk gene family members not contained in the
T66B region several cDNA clones were isolated from a testis cDNA
library constructed from male mice of the genotype
t.sup.w5/t.sup.w12, by hybridization with the probe
pCRt.sup.w5-155/170 derived from the T66Bk gene. Several cDNA
clones were isolated. All of them have a high sequence similarity
to T66Bk or T66Bk-2.
[0225] One of them, T66k-8 (FIG. 7b) is almost identical in
sequence to T66Bk-2 as far as sequence is available for both genes,
except that it contains a single base deletion leading to an
alteration of the ORF C-terminally to the protein kinase domain.
From the high sequence conservation of T66k-8 to T66Bk-2 it seems
not unlikely that T66k-8 is derived from the T66Bk-2 locus.
However, it is not clear how the single base change was introduced
into the cDNA clone, whether by a mistake in the RNA transcription,
processing, reverse transcription, or by another mechanism. For
instance, it has been shown that RNA editing resulting in a change
of the nucleotide sequence which can alter the ORF, can occur in
lower and higher eukaryotes. At the moment, such a mechanism cannot
be excluded as the cause of the observed alteration. Nor can it be
excluded that T66k-8 derives from a duplicated T66Bk-2 locus.
Alternatively, T66k-8 might be -derived from the t.sup.w5 allele of
T66Bk-2. Another cDNA was found that also contains a single base
deletion at a similar position as T66k-8. The genomic location of
the corresponding gene has not been determined. The alteration
predicted for the C-terminal tail of either gene product would be
expected to result in a change of the regulation and/or level of
their protein kinase activity and/or of the location of the protein
within the cell.
[0226] Another cDNA clone, T66k-7as (FIG. 7c), also isolated from
the cDNA library, has a very intriguing sequence and structure. It
contains a sequence strongly related to T66Bk/T66Bk-2, including
intron sequences from either side of the exon containing the single
long ORF and additional sequences from further downstream, inserted
in antisense orientation in the plasmid cDNA vector. Therefore
T66k-7as must be derived from an antisense transcript of a T66Bk
family gene. The predicted T66k-7as transcript does not contain a
long ORF. The intron sequence 5' to the ORF encoding exon of
T66Bk/T66Bk-2 is very A/T rich in antisense direction and
apparently serves as transcription stop and polyadenylation signal
during the synthesis of this antisense transcript. The sequences
contained in the BamHI fragment B9.1 of cat.15 which are related by
sequence to the 5'end of T66k-7as map to the vicinity of the
promoter of T66Bk suggesting that the promoter region of T66Bk
might contain elements controlling in cis the transcription of
T66Bk sense RNA as well as the transcription of T66Bk-2 antisense
RNA. If that were the case, antisense transcription might be
achieved by the same cis-control elements and thus occur at the
same stage as sense-RNA transcription. So far, no antisense
transcript coming from that locus of the T66B region was
identified. Nonetheless, the similarity of the structure and
sequence of T66Bk-7as to the head-to-tail arrangement and sequence
of T66Bk-2/T66Bk suggests that the T66Bk-2 gene of the T66B region
might be transcribed in antisense direction. In addition, another
T66Bk locus must exist which is transcribed in antisense direction,
gave rise to the cDNA T66k-7as and might be located within the T66B
region.
[0227] It is obvious that the expression of antisense transcript
complementary to mRNA transcribed from members of the T66Bk gene
family would be well suited to diminish the level of functional
gene products derived from that gene family. This could influence
the spermatozoa in two ways. If the antisense transcripts act in
both types of spermatids, those carrying the t-Responder and those
not carrying it, the former might be protected from that negative
action of antisense transcripts by a higher activity of its T66Bk
family gene products whereas the latter are not. In the
alternative, more likely way the antisense RNA transcripts might be
restricted to the former spermatids and lower the expression of
T66Bk gene products expressed in them. This would help to protect
the former from the negative action of hypermorphic Distorter gene
products, whereas the latter would be "poisoned" by them. This
"poisoning" would be caused by hyperactivation of the
Responder/Distorter signaling cascade.
[0228] Antisense RNA derived from (a) T66Bk family member(s) would
be expected to attenuate the negative effect of the Distorters and,
in that way is envisaged to contribute to the transmission ratio
distortion phenotype.
[0229] Another cDNA clone, T66k-20, isolated from the
t.sup.w5/t.sup.w12 testis cDNA library encodes yet another member
of the T66Bk gene family (FIG. 7d). Its ORF differs from T66Bk and
T66Bk-2 in a number of amino acid residues and in particular at the
N-terminal end which is 20 residues longer than that of T66Bk and
T66Bk-2 (FIG. 7e). Most likely, T66k-20 is derived from a gene
located in the T66A region, and thus may provide wild type
Responder activity.
[0230] The analysis of the transmission ratios of t.sup.lowH or
t.sup.low3H heterozygous with t.sup.h51t.sup.h18 by Lyon (1984),
showed a strong difference between the transmission ratio of
t.sup.lowH and t.sup.low3H. In addition, neither t-haplotype
reached the high value of a complete t-haplotype heterozygous with
a wild type chromosome. These data suggest the involvement of
several loci in the t-Responder function. At the present level of
analysis it is speculated that T66Bk, T66Bk-2, T66k-8, T66k-20 and
T66k-7as may cooperatively contribute to the t-Responder
function.
[0231] The testis cDNA library prepared from RNA of a male carrying
the t-haplotypes t.sup.w5/t.sup.w12 did not contain a cDNA clone
derived from the T66Bk gene. Therefore another testis cDNA library
was constructed from RNA of a male carrying the t-haplotypes
t.sup.6/t.sup.w5. Four clones containing a fragment of the size
expected from PCR amplification with the primer pair 161/144 were
identified and one of them was purified and sequenced (FIG. 9). The
sequence is identical to that of the cDNA pCRt.sup.h2-161/144 (FIG.
4a) in the region of overlap and extends it at the 5' as well as
the 3'-end. It is worth noting that the sequence ends in an intron
of the rsk3 locus in the T66B region and has no consensus
polyadenylation signal suggesting that the cDNA is not derived from
a properly processed mRNA molecule, but from a, possibly rare,
transcript which has not been spliced completely and may contain a
dA-rich intron sequence. This finding leaves open the possibility
that the T66Bk gene transcript might include the complete rsk3
locus in T66B from bp 438 of the coding region to the 3'-end.
[0232] In addition to the T66Bk family members encoded in the
t-haplotype, three more family members derived from the wild type
inbred strains Balb/c, C3H/N and 129/Sv were isolated either by
RT-PCR or on a genomic clone (FIG. 10). Again, high sequence
conservation to the t-haplotype family members was observed. The
gene pCR.Balb-66k has the same feature as the gene T66k-20, namely
a potential translation start site upstream of the one utilized by
T66Bk coding for additional 20 amino acid residues. It is not
clear, however, whether this translation start is efficiently used
since it does not conform with Kozak's rules demanding an A or a G
at position -3 upstream of the ATG codon.
[0233] In contrast, the genes pCR.C3H-66k and p.lambda.. 129-66k
differ significantly from all other T66Bk family members at their
C-terminus. Both genes contain a translation stop codon at triplet
position 434 resulting in a truncated protein of only 433 amino
acid residues whereas the remaining nucleic acid sequence is not
significantly different from those of the other members. The
truncation occurs outside the kinase domain suggesting that the
protein migth still be able to function as a kinase. However, the
alteration of the C-terminus might influence the regulation and/or
level of kinase activity. In this context it is interesting to note
that on the C3H background t-haplotypes are transmitted at a very
high ratio, whereas e.g. t.sup.o is transmitted at a reduced ratio
from males carrying the T/t-complex from Balb/c compared to the
ratio obtained by males of the genotype t.sup.o/C3H (Bennett et al.
1983). The 129Sv background also enhances the transmission ratio of
t-haplotypes similar to C3H (our observations). The shortened ORFs
in pCR.C3H-66k and p.lambda.. 129-66k might have an influence on
this behaviour. On the other hand, other T66Bk family members
encoding proteins of the same length as T66Bk might exist in these
strains in addition to the ones shown here.
[0234] Therefore, and in general, it is to be noted that the
genetic background of the animal strain involved may significantly
contribute to the expression of the phenotype in terms of the level
of distortion of the transmission ratio.
EXAMPLE 6
Transmission Ratio Distortion in Males Carrying Transgene
Insertions Encoding the T66Bk Kinase
[0235] To prove the involvement of T66Bk in the Responder phenotype
transgene constructs were made expressing the kinase gene T66Bk
(FIG. 4a) either under control of the testis promoter of c-kit
(tg4-3; tg4-13) or of the putative endogenous promoter of T66Bk
(FIG. 11) in transgenic mice (tg5-43; tg5-25). Mice carrying the
trangene integration were mated to mice carrying either the
t-haplotype t.sup.h51-t.sup.h18 expressing the t-Distorters D1 and
D2 or the wild type chromosomes C57BL/6 or Ttf/+tf (Lyon 1984).
Males of the appropriate genotype were mated to NMRI outbred
females and their offspring tested for carriers of the transgene.
The expectation based on the experiments of Lyon (1984) was that,
if T66Bk encodes a protein involved in transmission ratio
distortion the t-Distorters should enhance the transmission ratio
of the transgene, as is the case in the genotype
+t.sup.lowH+/t.sup.h51+t.sup.h18, whereas in males carrying wild
type chromosomes the transmission ratio of the transgene should be
lowered. Table 1 shows the data obtained so far. Interestingly, one
of the transgene integrations (tg4-3) must have occured on the Y
chromsome since it is only observed in males. In this case
offspring were examined for external sexual characteristics after
birth, the other transgene integrations were examined by PCR
analysis. The data demonstrate a significant distortion of the
transmission of the transgene confirming that T66Bk encodes
t-Responder activity. The data also demonstrate the potential of
the T66Bk gene in breeding strategies selecting for specific
genetic traits, in particular sex. In addition the data show the
usefulness of both promoters as control elements in achieving a
Responder phenotype.
[0236] However, the transmission distortion effect obtained is
considerably smaller than that observed with the genotype
+t.sup.lowH+/t.sup.h51+t.sup.h18 or +t.sup.lowH+/++tf (Lyon 1984).
This suggests that either the expression level of the T66Bk kinase
from the transgene constructs is not adequate or that the
expression of wild type Responder loci in spermatozoa carrying the
transgene diminishes the effect of the T66Bk gene. It should be
taken into consideration that the t.sup.lowH chromosome is carrying
loci selected by nature for an optimal effect on transmission ratio
distortion. In Lyon's analyses (1984) sperm carrying this
chromosome compete with sperm carrying either a wild type
chromosome or the t-Distorters t.sup.h51-t.sup.h18 probably in
combination with (a) wild type Responder locus (loci). In contrast,
the trangene integrations occurred outside of chromosome 17.
Therefore, transgene expression always occurs in sperm expressing
in addition (a) wild type Responder locus (loci). These sperm are
competing with sperm carrying either a wild type chromosome or the
t-Distorters t.sup.h51-t.sup.h18 probably in combination with (a)
wild type Responder locus (loci). The combination of T66Bk
expressed from the transgene with expression products from (a) wild
type Responder locus (loci) might be less effective in distorting
the transmission ratio than the combination of products expressed
by members of the T66Bk gene family, in particular T66Bk and
T66Bk-2, in the t.sup.lowH t-haplotype. Also, it has been
demonstrated that the genetic background has a considerable effect
on the ratio of transmission distortion achieved by various
t-haplotypes (Bennett et al., 1983). It is quite clear that the
expression level and/or activity of the T66Bk gene has to be
optimized in future experiments in order to obtain a stronger
transmission ratio distortion effect.
[0237] Also, control elements affecting the expression level such
as elements regulating transcription efficiency, transcript
processing and stability and translation efficiency, used for
transgene expression have to be optimised to achieve a maximal
effect. It would be convenient to select a tissue and stage
specific promoter such as the one controlling the expression of
T66k-20 preferably including its 5'-untranslated region, first
intron and 3'-untranslated region. Alternatively, an
3'-untranslated region known to increase the stability of the
corresponding mRNA could be used. We have noticed that transcripts
derived from T66k-20 are respresented at a high ratio in cDNA
isolated from a testis cDNA library constructed from RNA of mice
carrying t.sup.w5/t.sup.w12. In contrast, cDNAs derived from T66Bk
were not found and cDNAs derived from T66Bk-2 were highly
underrepresented, suggesting that the transcription level of
T66k-20 is considerably higher than that of the former loci.
[0238] However, transfer of this system for distortion of the
transmission of genetic traits, in particular of sex, to farm
animals might be achievable without a major effort since it is not
expected that amplification of T66Bk related genes also occurred in
farm animals which have not evolved transmission ratio distortion.
Therefore, T66Bk might have a much stronger effect on transmission
ratio when introduced into farm animals. The data presented here
open the prospect of producing farm animals fathering
preferentially or even exclusively offspring of the same sex, e.g.
only or predominantly females.
EXAMPLE 7
Cloning of Wild Type Members of the T66Bk Kinase Gene Family
[0239] The cDNAs pCR.Balb-66k and pCR.C3H-66k were isolated by
RT-PCR using the primer pairs 161/220 (220:
5'-CTTCCCCCTGGCTGGAC-3') from testis RNA of the inbred strain
Balb/c and C3H/N, respectively, cloned in the plasmid vector pCR2.1
(Invitrogen) and analyzed using the methods described in figure
legends 3 and 4. The extension step in the PCR was performed for 2
min. at 72.degree. C. The sequence of p.lambda.. 129-66k was
derived from an EcoRI subclone in pBluescriptKS made from a
lambda-FixII clone isolated from a genomic lambda-FixII library
using a cDNA fragment of T66Bk as probe. The lambda-library was
constructed from genomic DNA of the ES-cell line R1 (Nagy et al.
1993), according to the instructions of the supplier for the lambda
cloning and packaging kits (Stratagene). Library construction,
plating and screening by hybridization was according to standard
techniques (Sambrook et al. 1989) and the methods described in
figure legends 2, 3 and 4.
[0240] Primer sequences:
[0241] ACE
[0242] 5' GC CAA CCA GGG GAT A 3'; 5' CT GTC CGG TCA TAC TCT T
3'
[0243] c-kit
[0244] 5' CTT GTG TCC TTG GGA GAA 3'; 5' GGT GCC ATC CAC TTC AC
3'
[0245] mP1
[0246] 5' CGC AGC AAA AGC AGG AGC AG 3'; 5' CAT CGG ACG GTG GCA TTT
TT 3'
[0247] mouse rsk3
1 mouse rsk3 144: 5' TGG TCA AGC GAA AAT CTG TG 3' 145: 5' ATG GCC
TGG GGA TCA TCT AG 3' 146: 5' CAC CGC TTG CAC ACT GAG TA 3' cDNA
pCRt.sup.h2-161/144 155: 5' ATC GAT GTG TGG GGT CTT 3' 161: 5' GTT
TGG GAG GAG CTT GTG 3' 170: 5' CTA GTC GAG CCC TTG ATG 3' 181: 5'
TGG CAT CTT ATT GTC TAC 3' 191: 5' CCA AGC CCC TTT TTC TGA 3'
[0248] pSV-Sport1
[0249] seq5lib: 5' ATTTAGGTGACACTATAGAAGGTA 3'
[0250] Oligonucleotide sequences:
2 232: 5' CCC CCT TTA TCT GAC 3' 237: 5' TAT GCT GGC AGC ATC AAA
3'
[0251]
3TABLE 1 tg4 males genotype # female # male % male 4-3/5
th51-th18/C57BL 42 71 62.8 4-3/36 th51-th18/C57BL 33 55 62.5 4-3/39
th51-th18/C57BL 50 67 57.2 total: 125 193 60.7% 4-3/37 +tf/C57BL 42
29 40.8 4-3/187 C57BL/C57BL 52 37 41.6 total: 94 66 41.2% tg4 males
genotype # -tg # +tg % tg 4-13/80 th51-th18/C57BL 41 58 58.6
4-13/86 th51-th18/C57BL 45 55 55 4-13/97 th51-th18/C57BL 44 56 56
total: 130 169 56.5% 4-13/53 +tf/C57BL 56 47 45.6 4-13/96 +tf/C57BL
70 67 48.9 4-13/100 +tf/C57BL 53 47 47 total: 179 161 47.3% tg5
males genotype # -tg # +tg % tg 5-43/100 th51-th18/C57BL 13 29 69.0
5-43/101 th51-th18/C57BL 12 16 57.1 5-43/104 th51-th18/C57BL 26 28
51.8 5-43/105 th51-th18/C57BL 12 25 67.5 total: 63 98 60.8% 5-25/83
Ttf/C57BL 43 29 40.3 5-25/84 +tf/C57BL 37 24 39.3 total: 80 53
39.8%
[0252] Table 1:
[0253] Transmission ratio distortion in mice carrying transgenes
encoding the kinase gene T66Bk.
[0254] Two transgene constructs, tg4 and tg5 containing the protein
coding region of T66Bk were constructed in vitro and introduced
into the germ line by injection of DNA into one pronucleus of
fertilized eggs of the genotype
((C57BL/6.times.C3H/N)F1.times.C57BL/6) female X NMRI male and
retransfer of the zygotes or 2-cell embryos into NMRI foster
mothers. Male or female carriers of either transgene were mated to
mice carrying either the t-Distorters D1 and D2 on a single
t-haplotype chromosome (t.sup.h51-t.sup.h18) over Ttf, +tf or
C57BL/6, or either the wild type genotype Ttf/+tf or C57BL/C57BL.
Males carrying the appropriate genotype were identified by PCR
analysis and set up for test matings with NMRI outbred females. In
most cases, late embryonic stages were used as source of DNA for
testing individual offspring for the presence or absence of the
transgene, the remainder were tested using a tail piece as DNA
source. A chromosome 17 marker locus was tested in parallel to
control the quality of the DNA solution. The transgene tg4 of the
line 4-3 segregates with the Y-chromosome, suggesting that tg4 is
integrated on the Y chromosome. Therefore, in this case, offspring
were examined after birth for their sex using external sexual
characteristics. The breeding data demonstrate non-mendelian
inheritance of the transgene and, in the case of tg4-3, of sex. The
deviation from the expected 50% depends on the presence or absence
of t-Distorter loci, being significantly higher than 50% in the
presence and lower than 50% in the absence of t-Distorter loci, as
expected from the t-haplotype Responder locus Tcr. This confirms
the finding that T66Bk encodes t-Responder activity.
[0255] Methods:
[0256] Tg4 consists of the testis promoter of c-kit, base 45 to the
StyI site at base 683 (Rossi et al. 1992; Albanesi et al. 1996),
the cDNA t.sup.h2-161/144 and additional mouse rsk3 sequence
comprising bp 438 up to bp 998 of rsk3 (Kispert, 1990), and
IRES-.beta.geo containing the internal ribosome entry site IRES
(Ghaftas et al. 1991) and the .beta.gal-neo fusion gene and SV40
polyadenylation signal (Friedrich and Soriano 1991). In brief, the
testis promoter of c-kit was isolated by RT-PCR from testis RNA
using the primer pair 5'-ATGTAAGTGGCATGGAGT-3' and
5'-GCACACCGAAAATAAAA-3' and cloned into the plasmid vector pCR2.1
(Invitrogen). A NotI-BstEII fragment comprising the cDNA
t.sup.h2-161/144 from a vector NotI site at the 5'-end to a BstEII
site in the rsk3 homology region was ligated to NotI and BstEII
sites in the plasmid IRES-.beta.geo containing the rsk3 homology
region from the BstEII site to bp 998, 5' of the IRES-.beta.geo
gene. The 5'-end of the resulting construct containing an EcoRV
site from the vector pCR2.1 just 3' of the NotI site was replaced
by a NotI-StyI fragment containing the testis promoter of c-kit
cloned in the vector pCR2.1 by ligation of the NotI-StyI(blunt; the
StyI site was blunt-ended by treatment with the Klenow-fragment of
E.coli DNA polymerase I) fragment comprising bp 45 to bp 683 of the
c-kit promoter into the NotI and EcoRV sites of the construct. The
final transgene construct was released from the vector by digestion
with NotI and SaII.
[0257] Tg5 consists of 2637 bp (KpnI to PmII fragment) of the
genomic region upstream of the putative transcription start site of
T66Bk including most of the 5'-untranslated region and the putative
promoter of T66Bk (FIG. 11), the cDNA t.sup.h2-161/144 from the
HincII site (bp 293) to the EcoRI site in vector pCR2.1 including
the complete protein coding region and a HA-tag constructed into
the start site of translation, the IRES sequence and coding region
of human CD24 (Kay et al. 1991), and the modified intron and
polyadenylation signal of SV40-t (Huang and Gorman 1990). Tg5 was
constructed in several steps. First, an HA-tag encoding the peptide
sequence YPYDVPDYA was introduced at the translation start of the
cDNA t.sup.h2-161/144. Second, the putative promoter of T66Bk was
isolated as a 2.6 kb KpnI(blunt)-PmII fragment from the genomic
BamHI fragment B9.1 of cosmid cat.15, and ligated into EcoRV and
HincII sites of the vector containing the HA-tagged cDNA
t.sup.h2-161/144. The EcoRV site stems from the vector pCR2.1 while
the HincII site is contained in the 5'-untranslated region of the
cDNA t.sup.h2-161/144. In the third step the IRES sequence and
hCD24 coding sequence was cut as an EcoRI-EagI(blunted) fragment
from the plasmid pSLV-1, the modified intron and polyadenylation
signal of SV40-t were cut as a SnaBI-BamHI fragment from the Vector
pSV-Sport1 (Gibco/BRL), and both fragments were ligated together
into the previous construct opened at the vector sites EcoRI and
BamHI located at the 3'-end of the insert. The construction of an
HA-tag into the translation start site of T66Bk was done as
follows. First, two fragments of the cDNA t.sup.h2-161/144 were
amplified by PCR using the primer
5'-GGCGTAGTCTGGGACGTCGTATGGGTACATGTCAGAAAAAGG-3' and
5'-ATGTACCCATACGACGTCCCAGACTACGCCATGGAGAAATTTCAT-3', respectively,
in combination with the upstream primer 161 or the downstream
primer 188 (5'-ACCCTGGTTGTGGCAGTA-3'), respectively, creating an
overlapping region encoding the HA-tag sequence coding for the
peptide YPYDVPDYA, in frame with the translation start site of
T66Bk. The PCR was performed as described in figure legend 3 except
that 15 cycles were performed and 50 ng template were added. Then,
both fragments were isolated from an agarose gel and used as
template together in a second PCR. First 15 cycles of 30 sec.
94.degree. C., 2 min. 72.degree. C. were performed without primers,
the flanking primers 161 and 188 were added and a further 25 cycles
of 30 sec. 94.degree. C., 30 sec. 50.degree. C., 30 sec. 72.degree.
C. were performed. The resulting fragment containing the HA-tag
sequence was purified from an agarose gel, cut with HincII-EcoNI
and ligated in place of the HincII-EcoNI fragment of the original
cDNA clone t.sup.h2-161/144.
[0258] Testing of offspring for carriers of the transgene insertion
was done by first digesting a tissue sample of individual embryos
or mice in lysis buffer (100 mM Tris-HCl pH8.5/5 mM EDTA/0.2%
SDS/200 mM NaCl/200 .mu.g/ml Proteinase K) over night at 55.degree.
C., diluting an aliquot 20 fold in water followed by inactivation
of the Proteinase K by incubation at 80.degree. C. for 30 min., and
assaying 1 .mu.l in a 20 .mu.l PCR reaction as described in figure
legend 3 using the primer pair 309: 5'-CAGCCCATGAATCCATC-3' and
310: 5'-TGCCTTCGGTCTGAAAG-3' and the cycling conditions 2 min.
94.degree. C., 35 cycles 30 sec. 94.degree. C., 30 sec. 50.degree.
C., 1 min. 72.degree. C. A control PCR reaction assaying for the
genotype at the locus Hba-4ps in the distal region of the mouse
T/t-complex was performed where appropriate using the primer pair
Hb.1/Hb.2 and conditions as published (Schimenti and Hammer 1990).
This PCR reaction was also used to test for the presence of the
distal t-haplotype region t.sup.h18 containing the t-Distorter D2.
Likewise, presence of the proximal t-Distorter D1 in the
t-haplotype t.sup.h51 was assayed by testing for the presence of a
t-specific fragment at the Tcp1 locus. This was done by PCR using
the primer pair 5'-AGGAAAGCTTGCCCAAGAGA- ATAGTTAATGC-3' and
5'-AGGCGAATTCCATATCATCMTGCCACCAG-3'. The cycling conditions were 40
sec. 94.degree. C., 40 sec. 60.degree. C., 1 min. 30 sec.
72.degree. C., 35 cycles. Different wild type alleles at the locus
D17Mit46 from the middle of the T/t-complex were distinguished by
PCR using the primers Left: 5'-TCCACCCCACTACCTGACTC-3' and Right:
5'-CCCTTCTGATGACCACAGGT-3'. Cycling conditions were 40 sec.
94.degree. C., 40 sec. 50.degree. C., 40 sec. 72.degree. C., 35
cycles. This marker allows to distinguish between the allelic
variants of the strains C57BL/6, NMRI and Ttf/+tf.
[0259] All cloning procedures were performed according to standard
techniques (Sambrook et al. 1989), the production of transgenic
mice was done according to the methods described in Methods in
Enzymology, Vol. 225, Guides to Techniques in Mouse Development,
1993 (ed. P. M. Wassarman and M. L. DePamphilis). Mice carrying the
t-haplotype t.sup.h51-t.sup.h18 were obtained from Dr. M. F. Lyon
(Harwell, England), mice with the genotype Ttf/+tf were a gift of
Dr. K. Artzt (Austin, Tex.).
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* * * * *