The role of DNA modification in the maintenance of mammalian X-chromosome inactivation was investigated by using the technique of DNA transformation in mammalian cells. The ability of inactive X-chromosome DNA from adult mouse tissues to act in transformation for the X-linked hypoxanthine phosphoribosyltransferase gene (Hprt) could be ascertained by utilizing a recently discovered electrophoretic variant form of the hypoxanthine phosphoribosyltransferase enzyme and a previously available X: autosome translocation. Our findings indicate that inactive X-chromosome DNA from several tissues of adult female mice is strikingly inefficient, in comparison to active Xchromosome DNA, in eliciting genetic transformation for hypoxanthine phosphoribosyltransferase. These results provide in vivo evidence that is consistent with DNA modification playing an important role in the maintenance of X-chromosome inactivation.The somatic cells of normal diploid female mammals have two X chromosomes but only one X chromosome is active in each cell (1). The available evidence suggests that the single-active X is derived for most tissues by a process ofrandom inactivation of either the paternal or maternal X and that once the process is initiated the inactivated state of that X chromosome is maintained through successive cell divisions (2-7). The mechanism(s) responsible for maintaining a difference between the active and inactive states could involve theoretically alterations in the structure of DNA itself (8-10), chromatin structure (11), or chromosomal proteins (12, 13), or any combination thereof.Experimental attempts to reactivate the inactive X chromosome have been employed to provide a basis for defining the molecular mechanisms that are responsible for maintaining the inactive X. In general, attempts to select for reactivation of the X-linked hypoxanthine phosphoribosyltransferase gene (Hprt) suggest that random reactivation is a relatively rare event (5, 6). Recently, reactivation of human X-chromosome genes has been observed at relatively high frequency after 5-azacytidine treatment of human-mouse somatic cell hybrids containing an inactive human X chromosome (14-16). These findings suggest that DNA methylation may play a significant role in the maintenance of the inactive X.The role of DNA modification in the maintenance of the inactive X has also been examined by asking whether active and inactive X chromosomal DNA could function in DNA-mediated gene transfer (17). In brief, DNA was extracted from mutant cell lines carrying a defective Hprt gene on the active X chromosome and retaining an intact, inactive X chromosome. The DNA from these cells did not produce transformants that express hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), whereas control cells with a functional Hprt gene did. These authors argued that the failure ofsuch DNA to function in HPRT transformation implied a difference between inactive and active X-chromosome DNA. It is important to note that they assumed but could not prove that the...
We used a convenient quantitative dot blot assay to measure transcript levels for two X chromosomelinked genes, myo-2 and act4, in the nematode Caenorhabditis elegans. We show that there is dosage compensation of transcript levels for these two genes between XX herm.aphrodites and XO males and that a mutation in the dpy-21 gene, postulated from genetic analysis to be involved in control of X chromosome expression, can affect these transcript levels in the manner predicted. However, we observe the dpy-21 effects only at some stages of the life cycle and not at others. These results are generally consistent with earlier genetic and molecular evidence.In many animals one sex has two X chromosomes and the other sex only one. Despite this 2-fold difference in X chromosome dosage, most X-linked mutations cause similar mutant phenotypes in both sexes (1), and both sexes show equivalent levels of activities for many enzymes encoded by X-linked genes (2, 3). The mechanism of X chromosome dosage compensation responsible for this equivalence varies among different organisms. In mammals, it is accomplished by inactivation of one of the two X chromosomes in females during most of the life cycle (reviewed in ref. 2). In the fruit fly Drosophila, both X chromosomes are expressed in XX females, and compensation occurs by hyperactivation (relative to the autosomes) of the single X in XY males (reviewed in ref.3).The nematode Caenorhabditis elegans has two sexes: self-fertilizing hermaphrodites, which have two X chromosomes (XX), and males, which have one X chromosome (X0). There is no Y chromosome; the primary signal for sex determination is the X/A ratio, the ratio of X chromosomes to sets of autosomes (4). Early evidence for dosage compensation in C. elegans was genetic, based on analysis of hypomorphic mutations in X-linked genes. These are mutations causing partial loss of function such that the resulting phenotype varies with the level of the mutant gene product. Hypomorphic mutations of X-linked genes in C. elegans are observed to cause similar phenotypes in XX and X0 animals, indicating similar levels of the corresponding gene products, and thereby providing evidence for some mechanism of compensation for the difference in X chromosome dosage (5,6).To achieve dosage compensation, the level of X chromosome expression must presumably be dictated by the X/A ratio, but the genetic basis for this regulation is still unclear. At least four autosomal and two X-linked loci have been identified as possible-regulators of X chromosome expression. The phenotypes resulting from mutations in these genes are dependent on the X/A ratio in the mutant animal (7). These mutations result in the short phenotype known as dumpy (Dpy). Such X/A-dependent dpy genes can be grouped into two classes. Genes in the first class, all autosomal, are dpy-21 V (7), dpy-26 IV (7), dpy-27III (8), and dpy-28 III (9). Recessive mutations in dpy-21 and dpy-26 cause an increase in X expression based on the observation that they suppress the phenotypes re...
An electrophoretic variation for hypoxanthine phosphoribosyltransferase, HPRT, has been identified in samples of Mus spretus, a field mouse from southern Europe and in M. m. castaneus, a house mouse from southeast Asia. These mice will interbreed with laboratory mice to produce viable, fertile F1 progeny. The variation for HPRT segregates as an X chromosome gene in F1 and backcross progeny. Linkage analysis involving the markers Pgk-1 and Ags indicated a gene order of centromere—Hprt—Pgk-1—Ags in crosses involving both stocks of wild mice.
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