Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-07-26
2003-11-04
Wehbe′, Anne M. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S069100, C435S325000, C435S455000, C424S093100
Reexamination Certificate
active
06642369
ABSTRACT:
The present application is a U.S. National Stage Application of PCT/EP 98/07395, filed Nov. 18, 1998. This application also claims the benefit under 35 U.S.C. §119 of foreign application nos. EP 97 12 0190.0, filed Nov. 18, 1997 and EP 98 10 3596.7 (Mar. 2, 1998).
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 contraceptive.
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
t
)(Lyon 1984). Two more Distorters have been postulated by other authors (Silver and Remis 1987).
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
t
), leading to distortion in favor of R
t
. R
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
t
is transmitted at non-mendelian ratio to the offspring. If D2 or all the Distorters are present, the chromosome containing R
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
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
t
. The cis-action of R
t
suggests that R
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
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
).
Genetic mapping of molecular markers on partial t-haplotypes allowed a more or less precise localization of D1, D2, D3 and R
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
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
h44
and t
h51
, but is present in the partial t-haplotypes t
low
, t
h2
, t
h49
, t
6
, and in the complete t-haplotypes, e.g. t
w5
or t
w12
(FIG.
1
). Another partial t-haplotype, t
w71Jr1
(abbr. t
Jr1
) contains T66A and a part of T66B. The chromosomes t
h44
, t
h51
and t
Jr1
do not contain the R
t
function, whereas the other partial and complete t-haplotypes named above do. The t-haplotypes containing R
t
function must have the t-form of R, whereas the haplotypes t
h44
, t
h51
and t
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).
An extensive and careful search of this region for genes expressed during spermatogenesis has yielded a single gene, T66B-a or Tcp-10b
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
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
t
has been claimed to represent the candidate for R
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).
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.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
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
FIGS. 4
a
and
b
or
9
a
and
b
. 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.
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
(a) a nucleic acid molecule comprising the nucleic acid molecule as shown in
FIGS. 4
a
and
b
or
9
a
and
b
,
7
a
and
b
,
7
c, d
, and
e
,
7
f, g
, and
h
,
7
i, j
, and
k
,
7
l
or a fragment thereof;
(b) a nucleic acid molecule being an allelic variant or a homologue of the nucleic acid sequence of (a);
(c) a nucleic acid molecule hybridizi
Herrmann Bernhard
Kispert Andreas
Koschorz Birgit
Li Janice
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e. V.
Mueting, Raasch & Gebhardt, P. A.
Wehbe′ Anne M.
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