X inactivation Xplained

Random inactivation of one of the two female X chromosomes establishes dosage compensation between XY males and XX females in placental mammals. X inactivation is controlled by the X inactivation center (Xic). Recent advances in genome sequencing show that the Xic has evolved from an ancestral vertebrate gene cluster in placental mammals and has undergone separate rearrangements in marsupials. The Xic ensures that all but one X chromosome per diploid genome are inactivated. Which chromosome remains active is randomly chosen. Pairing of Xic loci on the two X chromosomes and alternate states of the X chromosomes before inactivation have recently been implicated in the mechanism of random choice. Chromosome-wide silencing is then initiated by the noncoding Xist RNA, which evolved with the mammalian Xic and covers the inactive X chromosome.     

Distribution of cortisol among its free and protein-bound fractions in rainbow trout (Oncorhynchus mykiss): evidence of control by sexual maturation.

1. Total cortisol concentrations did not differ in sexually matured female, male, and immature rainbow trout. 2. The per cent cortisol bound to a corticosteroid binding protein was greater in mature female fish (48.2%) than in mature male (16.0%) and immature fish (19.5%). 3. The mature female fish exhibited a lower percentage of free cortisol (21.8%) compared to mature males (44.8%) and immature fish (43.2%). 4. Parallel aspects of the teleostean cortisol binding-protein and the mammalian counterpart are compared and commented upon.     

Receptor-mediated endocytosis in Xenopus oocytes. I. Characterization of the vitellogenin receptor system

We have investigated the vitellogenin (VTG) receptor system in Xenopus oocytes since these cells are specialized for endocytosis. Oocytes have between 0.2 and 3 X 10(11) receptors per 1-mm cell. There is only a single class of receptors of low affinity (1.3 X 10(-6) M at 22 degrees C and 2-4 X 10(-6) M at 0 degree C), but high specificity (less than 5% nonspecific binding at 2 X 10(-6) M). The specific internalization rate of the VTG receptor (around 2 X 10(-3) s-1) is first order, highly variable, and at the upper end of the range of values reported for mammalian cells. The receptor association rate constant (9.6 X 10(2) M-1 s-1) is extremely low although the dissociation rate constant was immeasurable. Calcium is required for VTG binding, and low pH does not dissociate the VTG-receptor complex. Monensin treatment at 100 microM caused the loss of surface receptors with a t1/2 of 3 h and the accumulation of internalized ligand in a "pre-lysosomal" endocytic compartment. Conversely, the recovery of surface VTG receptors that were removed with trypsin occurred with a t1/2 of about 2 h. These observations indicate that oocytes have very large intracellular pools of receptors and that although surface receptors are internalized on the time scale of minutes, the intracellular pool is recycled on the time scale of hours.     

Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.     

X-changing information on X inactivation

In female somatic cells of mammalian species one X chromosome is inactivated to ensure dosage equality of X-encoded genes between females and males, during development and adulthood. X chromosome inactivation (XCI) involves various epigenetic mechanisms, including RNA mediated gene silencing in cis, DNA methylation, and changes in chromatin modifications and composition. XCI therefore provides an attractive paradigm to study epigenetic gene regulation in a more general context. The XCI process starts with counting of the number of X chromosomes present in a nucleus, and initiation of XCI follows if this number exceeds one per diploid genome. Recently, X-encoded RNF12 has been identified as a dose-dependent activator of XCI. In addition, other factors, including the pluripotency factors OCT4, SOX2 and Nanog, have been implicated to play a role in suppression of initiation of XCI. In this review, we highlight and explain these new and old findings in the context of a stochastic model for X chromosome counting and XCI initiation.     

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.     

Reactions of OH radicals with poly(U) in deoxygenated solutions: sites of OH radical attack and the kinetics of base release

Pulse radiolysis of N2O-saturated solutions of poly(U) in the presence of tetranitromethane showed that 81 per cent of the radicals formed are reducing in nature. Using data from other sources it has been estimated that 70 per cent of the OH radicals add to the base at C(5) and 23 per cent at C(6) while only 7 per cent abstract an H-atom from the sugar moiety. To a large extent the C(5) OH adduct radicals attack the sugar moiety of poly(U) thereby inducing strand breakage and base release. G (base release) = 2.9 can be subdivided into three components: (a) immediate (20 per cent), (b) fast (50 per cent) and (c) slow (30 per cent). The immediate base release must occur either during the free-radical stage or as a result of the rapid (t1/2 less than 4 min at 0 degree C) decomposition of a diamagnetic product. The fast and the slow processes are only readily observable at elevated temperatures, e.g. at 50 degrees C the half lives are 83 min and 26 h, respectively (Ea (fast) = 68 kJ mol-1, Ea (slow) = 89 kJ mol-1, A (fast) = 1.5 X 10(7) s-1, A (slow) = 1.9 X 10(9) s-1. It is concluded that there are three different types of sugar lesions giving rise to base release, structures for which are tentatively proposed.     

Characterization of triton X 100 extracted colipase from porcine pancreas

Colipase was isolated from porcine pancreas homogenate prepared in the presence of detergent (Triton X 100). After precipitation by ammonium sulfate and ethanol, the cofactor was purified by chromatography on SP-Sephadex in the presence of Triton X 100 and on DEAE-cellulose in the absence of detergent. Two molecular forms of porcine colipase were obtained. They represent 80 per cent (colipase A) and 20 per cent (colipase B), respectively, of the total colipase. Valine is the N-terminal residue of both proteins. Their aminoacid composition is similar to that found by Borgstrom for the two forms of porcine colipase. Determination of the sequence of the first sixteen residues at the N-terminal end of colipase A indicates that the cofactor undergoes no proteolytic degradation in this region of the molecule when extraction is carried out in the presence of detergent. The recovery of colipase is about 30 per cent.     

Characterization of Triton X 100 extracted colipase from porcine pancreas

Colipase was isolated from porcine pancreas homogenate prepared in the presence of detergent (Triton X 100). After precipitation by ammonium sulfate and ethanol, the cofactor was purified by chromatography on SP-Sephadex in the presence of Triton X 100 and on DEAE-cellulose in the absence of detergent. Two molecular forms of porcine colipase were obtained. They represent 80 per cent (colipase A) and 20 per cent (colipase B), respectively, of the total colipase. Valine is the N-terminal residue of both proteins. Their aminoacid composition is similar to that found by Borgstrom for the two forms of porcine colipase. Determination of the sequence of the first sixteen residues at the N-terminal end of colipase A indicates that the cofactor undergoes no proteolytic degradation in this region of the molecule when extraction is carried out in the presence of detergent. The recovery of colipase is about 30 per cent.     

Identification with G and R banding of the position of breakage points induced in human chromosomes by in vitro x-irradiation.

The aberrations seen in chromosomes of human peripheral-blood lymphocytes, X-irradiated in vitro, have been analysed in three types of preparations, treated to give G-banding; R-banding; and G-banding followed by R-banding on the same cells. The data from cells subjected to both banding techniques reveals that 30 per cent of the sites of chromosome breakage are situated at he interfaces between dark- and - light-stained bands. The results of all the analyses show that approximately 30 per cent of all breaks were located in either the telomere (19-5 per cent) or centromere (11-3 per cent) regions. Chromosomes rich in R-band material were not preferentially damaged, but chromosomes 12, 15, and particularly 17, were involved in aberrations more frequently than would be expected on the basis of their length. No breaks were found on the Y chromosome in the 114 male cells analysed, but the X did not appear to be spared from damage either in the male cells analysed, but the X did not appear to be spared from damage either in the male cell or the 136 female cells analysed. G and/or R-banding enables a much more accurate analysis of aberrations than can be obtained from the use of conventional staining techniques, and with these methods, it is shown that the numbers of induced asymmetrical and symmetrical exchanges are similar.     

Reduction of lumichrome by the radical anions of CO2 and lipoamide

The uptake of reducing equivalents of X CO-2 by lumichrome in spectrophotometric titrations has been re-examined in the light of a recently reported extinction coefficient of 10 500 M-1 cm-1 at pH 6, which is in agreement with 10 270 +/- 100 M-1 cm-1 determined here. The average uptake was 1.8 +/- 0.1, independent of pH in the range 6.3-9.0. The major product appears to be a dihydro-alloxazine, which can be reoxidized quantitatively to lumichrome by X Br-2 radicals or by O2. As in the case of dihydroflavins, oxidation by O2 is biphasic. As in the case of flavins, a two electron reduction of lumichrome was also observed with the disulphide monoanion of lipoamide (LS X 2-), but that reduction does not go to 100 per cent yield. Contrary to our earlier conclusions, which were based on an erroneous extinction coefficient, the combination of lumichrome radicals (2 X LcH----HLc--LcH) was of relatively little (less than or equal to 20 per cent) importance, and the behaviour of lumichrome on treatment with reducing species was rather similar to that of flavins.     

Targeted mutagenesis of Tsix leads to nonrandom X inactivation.

During X inactivation, mammalian female cells make the selection of one active and one inactive X chromosome. X chromosome choice occurs randomly and results in Xist upregulation on the inactive X. We have hypothesized that the antisense gene, Tsix, controls Xist expression. Here, we create a targeted deletion of Tsix in female and male mouse cells. Despite a deficiency of Tsix RNA, X chromosome counting remains intact: female cells still inactivate one X, while male cells block X inactivation. However, heterozygous female cells show skewed Xist expression and primary nonrandom inactivation of the mutant X. The ability of the mutant X to block Xist accumulation is compromised. We conclude that Tsix regulates Xist in cis and determines X chromosome choice without affecting silencing. Therefore, counting, choice, and silencing are genetically separable. Contrasting effects in XX and XY cells argue that negative and positive factors are involved in choosing active and inactive Xs.     

Properties of sex steroid-binding protein from Xenopus laevis blood serum and detection of an estradiol-binding component in frog liver cytosol which differs from sex steroid-binding protein]

Binding and physico-chemical properties of sex steroid-binding protein (SBP) from blood serum and those of estrogen-binding components from liver cytosol of pubertal male and female species of clawed frog Xenopus laevis were studied. It was shown that SBP from both sex species of X. laevis specifically binds estradiol (E2) (Ka=5 . 10(6) M-1). Concentration of SBP binding sites for E2 is 7 . 10(-12) mole per mg of protein. Testosterone 5alpha-dihydrotestosterone and E2 effectively compete with [3H]-E2 for SBP binding sites. Hexestrol, progesterone and corticosterone are weak competitors; estrone and E2-17-hemisuccinate do not compete at all. The Strokes radius of SBP is 4.4 nm; sedimentation coefficient is 4.6S. Molecular weight of SBP is 88000; f/f0 is 1.5 SBP from male frog sera has been purified 8.6-fold with 13% yield. Gel-filtration of [3H]-E2 complexes with liver cytosol proteins shows that the livers of male and female frog X. laevis consol proteins shows that the livers of male and female frog X. laevis contain very low amounts of macromolecular component, which specifically binds E2; this component differs from serum SBP in size and in hormonal specificity. It is assumed that this component is a receptor for estrogens.     

The Probability to Initiate X Chromosome Inactivation Is Determined by the X to Autosomal Ratio and X Chromosome Specific Allelic Properties

Background

In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X∶A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI.

Methodology/Principal Findings

To obtain more insight in the role of the X∶A ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X∶A ratios, provides evidence that the X∶A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X∶A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator.

Conclusions

The present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X∶A ratio. This finding supports the presence of an X-encoded activator of the XCI process.     

Modulation of P2X3 receptors by spider toxins

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.     

An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene

Early in female mammalian embryogenesis, one of the two X chromosomes is inactivated to compensate the gene dosage between males and females. One of the features of X chromosome inactivation (XCI) is the late replication of the inactivated X chromosome. This study reports the identification, by competitive PCR of nascent DNA, of a replication origin in intron 2 of the human X-linked HPRT gene, that is functional only on the inactive X. Features frequently associated with replication origins, including a peak of enhanced DNA flexibility, a perfect match to the yeast ACS sequence, a 14/15 match to the Drosophila topoisomerase II consensus, and a 20/21 match to an initiation region consensus sequence, were identified close to the replication origin. The origin is located approximately 2 kb upstream of a matrix attachment region (MAR) and also contains two A:T-rich elements, thought to facilitate DNA unwinding.     

Effect of particle size on the anthelmintic efficacy of mebendazole against Nippostrongylus brasiliensis in the rat.

The effect of reduction in particle size on the anthelmintic efficacy of mebendazole (methyl 5 (6)- benzoyle 1–2 benzimidazole carbamate) was studied in rats undergoing a primary infection with N. brasiliensis. A single oral treatment with fine ground mebendazole (particle size spectrum—54·95 per cent of particles less than 10·62 μ dia.; 86·06 per cent less than 21·27 μ) removed more than 98 per cent of adult worms from the intestine at a dose rate of 12·5 mg/kg body wt. On the other hand the best result achieved with coarse ground mebendazole (18·47 per cent of particles less than 10·62 μ dia; 42·26 per cent less than 21·27 μ dia) was 58 per cent of adult worms removed at a dose rate of 100 mg/kg body wt. It was also shown that fine ground mebendazole adversely affected migrating third stage larvae of N. brasiliensis.     

Fertility,sex determination,and the X chromosome

Because of its function, the X chromosome has a special status in mammalian genomes, with the specific occurrence of genes that influence both female and male fertility. Long ago, the XO karyotype (Turner syndrome) was associated with infertility, proving the correlation between normal X chromosome dosage and normal female fertility. Nevertheless, the search for specific X-borne fertility genes was not completely successful and suggested, instead, that female X-linked fertility, for example, depends upon groups of X-linked genes. Conversely, X-linked hyperfertility has been observed in sheep, where a mutation in BMP15 leads to a hyperfertile phenotype, but only in the heterozygous state. Many male fertility genes map to the X chromosome, consistent with a genetic model developed in the early 1990s. Ironically, NR0B1 (formerly DAX1), once presented as the paradigm of genes responsible for ovarian development and function, is probably one of these male fertility factors and is active in the maintenance of spermatogenesis. Indeed, duplications of this gene on the human X chromosome lead to XY sex reversal, as NR0B1 is able to counterbalance the effect in humans. Nevertheless, invalidation experiments in mice demonstrate the effect of this factor on male germ-cell production.