Nonrandom X-chromosome inactivation (XCI), also known as skewing, has been documented in the blood cells of a significant proportion of normal aging women by the use of methylation-based assays at the polymorphic human androgen receptor locus (HUMARA). Recent data obtained with a new transcription-based XCI determination method, termed suppressive polymerase chain reaction (PCR), has shed controversy over the validity of XCI ratio results obtained with HUMARA. To resolve this disparity, we analyzed XCI in polymorphonuclear leukocytes of a large cohort of women aged 43 to 100 years with the use of HUMARA (n ؍ 100), a TaqMan single nucleotide polymorphism (SNP) assay (n ؍ 90), and the suppressive polymerase chain reaction (PCR) assay (n ؍ 67). The 3 methods yielded similar skewing incidences (42%, 38%, and 40%, respectively), and highly concordant XCI ratios. This confirms that the skewing of XCI ratio seen in blood cells of aging women is a bona fide and robust biologic phenomenon. IntroductionGene dosage compensation between XX females and XY males occurs by the random inactivation of one of parental X chromosomes in the female embryo. 1 According to the Lyon hypothesis, 1 the fraction of cells with inactivation of each X chromosome should be equivalent. A deviation from the 1:1 X-chromosome inactivation (XCI) ratio is referred to as skewing. We have previously shown that skewing of XCI increases significantly in the blood cells of female subjects from the neonatal period to old age. 2 This observation has subsequently been confirmed by others 3-6 and has important implications for understanding the physiology of aging hematopoiesis and the pathogenesis of age-related hematopoietic malignancies. The concordance of skewing documented in elderly monozygotic versus dizygotic twins 7 supports a genetic component to the trait, and work by Abkowitz et al 8 in a feline model suggests an age-dependent hemizygous cell-selection process.A recent report 9 argued against the skewing phenomenon in blood cells of aging women. In this study, 9 the blood cells of 40 women older than 65 years of age were analyzed by both a methylation-based assay (ie, human androgen receptor locus [HUMARA]) and a novel real-time polymerase chain reaction (PCR) assay, termed suppressive PCR, in which allele-specific primers are used to identify coding single nucleotide polymorphisms (SNPs) in several X-linked genes, including the iduronate-2-sulphatase gene IDS. The use of HUMARA showed that 30% of women had skewed XCI in their blood cells, whereas the suppressive PCR assay did not show any significant skewing. 9 These observations led the authors to conclude that the skewed XCI patterns observed with HUMARA were not an accurate reflection of the proportion of cells bearing a particular X chromosome in the active state but rather reflected age-related dysregulation of methylation patterns. 9 Although this explanation is a plausible one for these data, it is at odds with previously published reports validating the HUMARA assay against trans...
Over the last 10 years considerable progress has been made in the immunological and biochemical characterization of oviduct-specific glycoproteins. It is now well established that a subclass of these secretory products, designated as oviductins, associate with the zona pellucida of the ovulated oocyte and with the early embryo. Recent reports on the cloning of cDNAs of oviductins from various species, including that of golden hamster (Mesocricetus auratus) oviductin by our laboratory, allowed us to compare their deduced amino acid sequences with those of other proteins. Optimal alignment analysis showed that oviductins contain regions of significant similarity with catalytically inactive mammalian members of the bacterial and microfilarial chitinase protein family. Most importantly, a close examination of the hamster and human deduced amino acid sequences revealed that both glycoproteins possess contiguous Ser/Thr rich repeated units, clustered in their carboxy-terminal portions. These mucin-type motifs are similar in the hamster and human glycoprotein, although hamster oviductin contains more of these complete units. This striking feature might indicate that these molecules play a similar role to mucin-type glycoproteins, e.g., in protecting the oocyte and early embryo against attacks from their environment. W e propose a model whereby oviductins are targeted to the oocyte via the interaction of their chitinase-like domains with specific oligosaccharide moieties of the zona pellucida. Once localized to this structure, oviductin molecules would act as a protective shield around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains. o 1995 Wiley-Liss, Inc.
The Cas-Br-E murine leukemia virus (MuLV) induces a degenerative myeloencephalopathy leading to hindlimb paralysis when inoculated into newborn mice. To map the viral DNA sequences encoding the determinant of neurological degeneration, we constructed chimeric viruses in vitro with parental genomes from Cas-Br-E MuLV and from nonparalytogenic MuLVs. We found that a 1.5-kilobase-pair env Cas-Br-E fragment was sufficient to confer the full paralysis-inducing potential to chimeric viruses. This region encodes the 19 carboxyl-terminal residues of the leader sequence, all of gp7O, and the 45 amino-terminal residues of the transmembrane protein (pl5E). Within this env region, we identified a 372-base-pair fragment which was necessary for the full paralysis-inducing potential of the virus and which influenced the development of the disease in a strain-dependent manner. This domain encodes the 19 carboxyl-terminal residues of the leader peptide and the first 67 amino-terminal residues of gp7O. We propose that Cas-Br-E MuLV induces spongiform degeneration through binding of its gp7O to a specific cellular receptor.The Cas-Br-E wild mouse ecotropic murine leukemia virus (MuLV) has been shown to induce a progressive form of hind-limb paralysis after inoculation into susceptible laboratory mice (for review, see refs. 1 and 2). This clinical syndrome is the consequence of a spongiform myeloencephalopathy predominantly involving the brainstem and the anterior horn of the lumbosacral region of the spinal cord (3-6). Using chimeric viruses constructed in vitro, we have previously mapped the primary determinant of this spongiform degeneration within a 3.9-kilobase-pair (kbp) pol-env fragment of the viral genome (7). Sequencing of this region revealed that the pol gene product and piSE diverged very little from homologous proteins of other nonparalytogenic MuLVs, while the Cas-Br-E gp7O sequence was found to diverge significantly from that of other nonparalytogenic MuLVs (8). We also showed that the long terminal repeat (LTR) region of the genome harbors a second determinant of pathogenicity influencing the incidence of the disease and determining in which region of the central nervous system (CNS) this virus induces the spongiform lesions (9).To determine more precisely which viral gene harbors the primary determinant of the spongiform lesions, we constructed additional chimeric viruses with DNA from parental paralytogenic Cas-Br-E and nonparalytogenic amphotropic, endogenous ecotropic, or Moloney MuLVs. We show here that chimeric viruses harboring only the env gene of Cas-Br-E MuLV genome induce spongiform lesions and remain paralytogenic. MATERIALS AND METHODSMice. SWR/J pregnant females were purchased from The Jackson Laboratory. SIM.S and NFS/N mice were from our breeding colony. The virus suspensions were filtered through a HAWP nitrocellulose filter (pore size, 0.45 um; Millipore) and injected intraperitoneally (0.15 ml) into newborn (<48 hr) mice.Cells and Viruses. The Cas-Br-E (pNE-8) molecularly cloned parental M...
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