Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-09-30
2003-10-14
Ungar, Susan (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06632934
ABSTRACT:
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns nucleic acid segments isolated from human and murine sources, which encode a male germ cell specific protein, designated MORC. Various methods for making and using MORC DNA segments, DNA segments encoding synthetically-modified MORC proteins, and native and synthetic MORC polypeptides are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications. Also disclosed are methods for identifying MORC-related polynucleotides and polypeptides, and methods for diagnosing and treating infertility or cancer, and in particular, testicular cancer, as well as screening methods for compounds that are involved with the development of cancer or spermatogenesis.
1.2 Description of Related Art
The genetic control of spermatogenesis is complex (Sassone-Corsi, 1997). Mutations at multiple loci and in structurally and functionally disparate genes in the mouse genome affect gametogenesis (Handel, 1987). Most mutations are pleiotropic, causing multi-system pathologies rather than isolated spermatogenic abnormalities. For example, the autosomal recessive mutation weaver, which results in degeneration of germ cells, also causes loss of the cerebellar granular cell layer and ataxia in affected mice (Vogelweid et al., 1993). The histologic phenotypes of mutations that affect germ cells are varied and include both reduced cell numbers and abnormal cell morphologies. Further complicating the understanding of germ cell biology is the fact that genes known to be essential for spermatogenesis participate in multiple cellular processes, including transcriptional control (Nantel et al., 1996; Blendy et al., 1996), cell proliferation (Toscani et al., 1997), protein folding (Dix et al., 1996) and DNA repair (Baker et al., 1995; Donehower et al., 1992).
1.2.1 Genetic Control of Mouse Spermatogenesis
The genetic control of mouse spermatogenesis has been extensively reviewed in the literature (Handel, 1987). Briefly, spermatogenesis is a complex and highly ordered developmental process, lasting 36 days in mice. Three phases of spermatogenesis can be distinguished: mitotic proliferation and renewal of spermatogonia, or stem cells; meiotic reduction division of spermatocytes; and differentiation of haploid spermatids into mature sperm cells, or spermiogenesis. The first meiotic division in protracted, with cells remaining in pachytene stage for 11 days. During this time, homologous chromosomes pair and recombine, and there is extensive DNA repair synthesis and transcription. Many genes must act during this stage of spermatogenesis, and it is the target of a number of mutations.
1.2.2 Mouse Spermatogenesis Mutations
A large number of spontaneous and induced mouse mutations resulting in abnormalities of normal spermatogenesis and fertility have been identified (Table 1). Recent reviews cataloging these mutations demonstrate the genetic heterogeneity and phenotypic pleiotropy of infertility (Handel, 1987; Chugg , 1989; Simoni, 1994; Wilmut et al., 1991). Mutations can be divided into three general groups. Pre-testicular phenotypes are the result of pituitary abnormalities or improper embryogenesis and germ cell migration (e.g., gcd). Intratesticular phenotypes result from mutations which manifest as abnormalities of the germ cells themselves (spermatogonia, spermatocytes, spermatids) (e.g., dazla). Post-testicular phenotypes encompass mutations that produce spermatozoa with abnormal function (e.g., hotfoot).
The majority of the mutations in Table 1 are pleiotropic, causing multisystem pathologies rather than isolated abnormalities involving spermatogenesis. For example, the autosomal recessive mutation sks (skeletal fusions with sterility) results in arrest of germ cell development at late meiotic prophase but also causes skeletal fusions of vertebrae and ribs, resulting in body shortening and tail kinks (Vogelweid et al., 1939). In many of these mutations, the effects on germ cells are broad and include both reduced cell numbers and abnormal cell morphologies. The genes associated with infertility have various and sundry functions and include metabolic proteins (ornithine decarboxylase), heat shock proteins/molecular chaperones proteins (Hsp70-2), transcriptional activators (CREM), and proteins involved in DNA repair and maintenance of genome stability, such as the DNA mismatch repair homologue pms2 and the p53 gene (Baker et al., 1995; Donehower et al., 1992).
1.2.3 Spermatogenesis and DNA Repair Mutations
Some of the best characterized genes required for spermatogenesis are those involved in DNA repair. DNA repair defects that delay or prevent the completion of meiotic recombination lead to disruption of the meiotic process (Baker et al., 1995; Arnheim and Shibata, 1997; Edelmann et al., 1996; Hawley and Friend, 1996; Kolodner, 1995; McKee, 1996; Modrich and Lahue, 1996; Rose and Holm, 1993), typically resulting in arrest at the pachytene stage. Factors have been identified which are exclusively required for meiotic events and others which play roles in both mitotic and meiotic cells. Topoisomerase II conditional mutants are one example of the latter. These mutants exhibit both enhanced mitotic recombination and meiotic pachytene arrest (Rose and Holm, 1993). A number of recent reviews have described how eukaryotes maintain chromosome integrity in meiotic cells through DNA repair (Kleckner, 1996; Stahl, 1996; Roeder, 1997). Meiotic cells seem to have developed mechanisms functionally equivalent to mitotic cell cycle checkpoints to sense DNA strand breaks and prevent cells from progressing through the cell cycle until DNA damage is resolved. In yeast it has been shown that several of the proteins involved in mitotic DNA strand-break cell cycle checkpoints (Rad17, Rad 24 and Mec1) are necessary for preventing cells from progressing into meiotic division I before recombination is complete (Handel, 1987). Based on these and similar observations, it has been proposed that the meiotic cell cycle is an evolutionary product of mitosis, with diploidy and strand break-sensing checkpoint mechanisms serving to ensure the integrity of the genome (Kleckner, 1996).
An interesting feature of mice missing various DNA repair components is that many targeted mutations that disrupt normal DNA repair and genome stability genes also have profound effects on germ cell development and spermatogenesis (Arnheim and Shibata, 1997)(Table 2). Some of these mutations involve defects in gametogenesis in both sexes (for example the mlh1 and Atm−/−mice), while deficiencies in other genes cause male germ cell arrest specifically (pms2−/−mice). Clearly male and female gametogenesis are biologically different processes although the molecular basis for the difference is not known.
TABLE 1
MOUSE SPERMATOGENESIS MUTATIONS
Female
Locust
a
Type
b
Fertile?
Germ Cell Phenotype
Other Phenotype
Ref
A-myb
KO
Yes
Lack of spermatids, post meiotic cells,
Mammary proliferation
[1]
degeneration of primary
defective
spermatocytes, apoptosis.
Bax (Bc1-2 partner)
KO
No
Multinucleate giant cell, cell death,
Lymphoid hyperplasia
[4]
no mature spermatids
bs (blind sterile)
AR
Yes
Acrosome absent in spermatids
bilenticular cataracts
[12]
c (albino)
AR
Yes
Reduced spermatids, abnormal sperm
pigmentation loss
[13]
in epidydimis
CREM (cyclic AMP
KO
Yes
Heterozygotes- reduced fertility.
Runting
[2, 14]
response element
Homozygotes- apoptosis, absence of
modulator
spermatids/spermatozoa
dazla
KO
No
Azoospermia, depletion of germ cells
None identified.
[15]
after mitotic proliferation
desert hedgehog
KO
Yes
Few spermatids on 129-C57BL/6J
None identified.
[16]
background, primary spermatocyte
degeneration on 129
gcd (germ cell
TKO
No
Albright George M.
Hess Karl D.
Inoue Norimitsu
Moreadith Randall W.
Watson Mark L.
Board of Regents , The University of Texas System
Ungar Susan
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