Genetic inactivation of the alpha-galactosidase locus in carriers of Fabry's disease.

Skin fibroblasts from a patient with Fabry's disease showed deficient activity of alpha-galactosidase. Fibroblasts from his mother and sister had two distinct clonal populations, one with enzymatic activity and the other enzyme deficient. This provides evidence of genetic inactivation at the alpha-galactosidase locus and makes possible the detection of carriers of Fabry's disease even when the enzymatic activity in their leukocytes and uncloned fibroblasts is within the range of controls.

Human-mouse somatic cell hybrids with single human chromosome (group E): link with thymidine kinase activity.

Mouse somatic cells lacking thymidine kinase were mixed in culture with human diploid cells lacking hypoxanthine guanine phosphoribosyl transferase, and hybrid cells were isolated and maintained in a selective medium containing hypoxanthine, aminopterin, and thymidine. The hybrid cells at the time of isolation had karyotypes consisting predominantly of mouse chromosomes but with one human chromosome, a submetacentric member of group E, apparently giving thymidine kinase to the hybrid cell. However, after long-term propagation in the selective medium this chromosome has been lost, although cells continue to show thymidine kinase activity as demonstrated by the incorporation of (3)H-thy-midine into DNA in the hybrid cell. The hybrid cells have only mouse electro-phoretic variants for glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase, suggesting that the human genetic loci for these enzymes are not represented in the hybrid genome and may be unlinked to that for thymidine kinase.

Interferon production and action in mouse, hamster and somatic hybrid mouse-hamster cells.

A hybrid mouse-hamster cell line was developed from a mouse cell line which produces a high titer of interferon and is sensitive to its action, and a hamster cell line which produces little interferon and is relatively insensitive to its action. Parental cell lines demonstrated complete species specificity with respect to interferon production and action. The hybrid cells produced interferon (or interferons) effective when tested on the mouse cell line and primary hamster cells; the hybrids were sensitive to the action of both mouse and hamster interferons. Hybrid cells produced ten times more hamster interferon than the parent hamster cell line and were eight times more sensitive to hamster interferon than the parent hamster cell line.

Human and mouse hypoxanthine-guanine phosphoribosyltransferase: dimers and tetramers.

Human and mouse hypoxanthine-guanine phosphoribosyltransferase subunits combine to form an active heteropolymer. Dimers form the basic subunit structure of the enzymes, yet the dimers can readily associate to form tetramers. The equilibrium between dimers and tetramers is significantly influenced by the ionic strength of the enzyme solvent.

Human thymidine kinase gene locus: assignment to chromosome 17 in a hybrid of man and mouse cells.

The human chromosome retained in a hybrid clone derived from human cells and a moluse line deficiemt in thymidine kinase has the quinacrinefluorescence pattern characteristic of chromosome 17.

X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: heterozygote has two clonal populations.

Clones of skin fibroblasts cultured from the mother of two sons with X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency (Lesch-Nyhan syndrome) were assayed for activity of this enzyme by measurement of the incorporation of (3)H-guanine into guanylic acid as counts per minute per microgram of protein and by autoradiography. The demonstration of two populations of clones, wild-type clones with normal enzyme activity and mutant clones unable to incorporate (3)H-guanine, is evidence that the locus for hypoxanthineguanine phosphoribosyl transferase on one of the X chromosomes is inactive.

X-chromosome inactivation: molecular mechanisms and genetic consequences.

Mammalian X-chromosome inactivation results in dosage compensation for X-linked genes. More than 30 years after its discovery, the molecular bases of this inactivation are being revealed. Multiple mechanisms are responsible for the initiation of this developmental event and the maintenance of the inactive state. Somatic cellular mosaicism, which is the genetic consequence of X-chromosome inactivation, has a profound influence on the phenotype of mammalian females.

Chromatin loop structure of the human X chromosome: relevance to X inactivation and CpG clusters.

Part of the higher-order structure of chromatin is achieved by constraining DNA in loops ranging in size from 30 to 100 kilobase pairs; these loops have been implicated in defining functional domains and replicons and possibly in facilitating transcription. Because the human active and inactive X chromosomes differ in transcriptional activity and replication, we looked for differences in their chromatin loop structures. Since the islands of CpG-rich DNA at the 5' ends of X-linked housekeeping genes are the regions where functional differences in DNA methylation and nuclease sensitivity are found, we looked for scaffold association of these sequences after extraction of histones with lithium diiodosalicylate. Specifically, we examined the 5' CpG islands within the hypoxanthine phosphoribosyltransferase, glucose 6-phosphate dehydrogenase, P3, GdX, phosphoglycerate kinase type 1, and alpha-galactosidase loci in human lymphoblasts obtained from individuals with 1 to 4 X chromosomes. Although we detected no scaffold-associated regions near these genes, we found several such regions at the ornithine transcarbamylase and blood clotting factor IX loci. Our results suggest that the CpG islands are excluded from the nuclear scaffold and that even though transcriptionally active, housekeeping genes are less likely than X-linked tissue-specific genes to be scaffold associated. In all cases, the pattern of scaffold association was the same for loci on active and inactive X chromosomes.

X chromosome inactivation: theme and variations.

My contribution to this special issue on Vertebrate Sex Chromosomes deals with the theme of X chromosome inactivation and its variations. I will argue that the single active X--characteristic of mammalian X dosage compensation--is unique to mammals, and that the major underlying mechanism(s) must be the same for most of them. The variable features reflect modifications that do not interfere with the basic theme. These variations were acquired during mammalian evolution--to solve special needs for imprinting and locking in the inactive state. Some of the adaptations reinforce the basic theme, and were needed because of species differences in the timing of interacting developmental events. Elucidating the molecular basis for the single active X requires that we distinguish the mechanisms essential for the basic theme from those responsible for its variations.

Concerning the role of X-inactivation and DNA methylation in fragile X syndrome.

Elucidation of the role of DNA methylation in X chromosome inactivation along with recent studies of the fragile X mutation suggests that DNA methylation is likely to be a late event in the pathogenesis of the fragile X syndrome. Thus far, the evidence does not support suggestions that an impediment to X reactivation and failure to demethylate the inactive X in oocytes is responsible for silencing the fragile X. The role of DNA methylation is probably secondary to amplification of the CGG repeat to a critical size whether on active or inactive X. Further studies are needed to determine if late replication of the inactive X predisposes the locus on that chromosome to more extensive amplification.

Severe phenotypes associated with inactive ring X chromosomes.

Mental retardation and congenital malformations in individuals with small ring X chromosomes are often due to the functional disomy that results from failure of these chromosomes to undergo X inactivation. Such chromosomes either lack the XIST locus or do not express it. We have carried out genetic analysis of the ring X chromosomes from two girls with a 45,X/46,X,r(X) karyotype, mental retardation, and a constellation of abnormalities characteristic of the severe phenotype due to X disomy. In each case the ring X chromosome included an intact XIST locus which was expressed; the breakpoints were distal to DXS128, and therefore outside the XIC region; transcription analysis of alleles at the androgen receptor locus confirmed that these were inactive chromosomes. The characteristics of the XIST RNA were similar to the wild-type. Additional studies in cultured fibroblasts showed a second ring in a small percentage of the cells. The association of severe phenotype with an inactive X chromosome most likely reflects the presence of a second ring X chromosome which was active at least in some tissues during embryogenesis, but is no longer prominent in the tissues we analyzed.

Some insights into X chromosome inactivation from studies of human cells.

The inactivation of all but one X chromosome in the somatic cells of mammalian females in an important mechanism for regulating X chromosomal genes. Although the molecular basis for the single active X remains to be elucidated, recent studies have provided some insights. Inactivation occurs about the time of implantation, at least in mouse embryos. On the other hand, germ cells from human embryos have two active X chromosomes and may never be subjected to inactivation. As a consequence of X inactivation, one X chromosome becomes the sole determinant of X specified characteristics of the cell, resulting in cellular mosaicism. Transfer of gene products between cells occurs via gap junctions and by pinocytosis so that in heterozygotes carrying X linked mutations, the mutant phenotype may be masked by the presence of cells or normal type. There is considerable evidence that inactivation in the embryo is random with respect to parental origin of the X chromosome, but that selection occurs subsequently, and may lead to elimination of a whole population of cells in the female. Although there is evidence for preferential inactivation of the paternal X in extra-embryonic membranes of rodents, studies in human embryos have not been confirmatory. Analysis of clonal populations from heterozygotes for X chromosome mutations have been used to determine whether a whole X chromosome is inactive or only parts thereof. Individuals with multiple X chromosomes often manifest somatic abnormalities as well as gonadal ones. There is recent evidence that some loci on the short arm may escape inactivation. X inactivation in diploid human cells has been shown to be a very stable process. The silent X, once inactivated, maintains inactivity despite attempts to turn it on. Our recent studies of enzyme activity in cells from a human triploid abortus (69, XXY) with two active chromosomes indicates that the "active chromosomes" are also stably maintained. The presence of two active chromosomes in triploid cells implicates the autosomes in the regulation of X chromosome activity.

Non-random X chromosome inactivation in mammalian cells.

A salient feature of mammalian X dosage compensation is that X-inactivation occurs without regard to the parental origin of either active or inactive X. However, there are variations on the theme of random inactivation, namely paternal X inactivation in marsupials and in placental tissues of some mammals. Whether inactivation is random or paternal seems to depend on the time when this developmental program is initiated. As deletions of the X inactivation center (XIC/Xic) and/or the X inactive specific transcript (XIST/Xist) gene result in failure of cis X-inactivation, mutations in genes from this region might lead to preferential inactivation of one X chromosome or the other. The Xce locus in the murine Xic is considered a prototype for this model. Recent studies suggest that choice involves maintaining the activity of one X, while the other(s) by default is programmed to become inactive. Also, choice resides within the XIC, so that mutations elsewhere, although perhaps able to interfere with cis inactivation, are not likely to affect the X chromosome from only one parent. Mutations affecting the choice of active X will be more difficult to detect in humans than in inbred laboratory mice because of the greater allelic differences between maternal and paternal X chromosomes; some of these differences predispose to growth competition between the mosaic cell populations. I suggest that the skewing of inactivation patterns observed in human females most often occurs after random X inactivation, and is due mainly to cell selection favoring alleles that provide a relative growth advantage.

Insights into X chromosome inactivation from studies of species variation, DNA methylation and replication, and vice versa.

I am indebted to Mary Lyon as her X-inactivation hypothesis stimulated my mentor, Barton Childs, and in turn, myself, to think about the consequences of X-inactivation in heterozygous females. I often reread her original papers setting forth the single active X hypothesis, and still marvel at the concise and compelling exposition of the hypothesis and the logical predictions which seemed prophetic at my first reading, and have survived the test of time. My contribution to this Festschrift reviews evidence derived from studies of DNA methylation, species variation and DNA replication that reveals an important role for methylated CpG islands and suggests a role for late DNA replication in propagating X inactivation from one cell to its progeny. These studies also show that X inactivation is a powerful research tool for identifying the factors which program and maintain developmental processes.

Glucose-6-phosphate dehydrogenase as a probe for the study of X-chromosome inactivation in hunan females.

The electrophoretic variants of G6PD have provided evidence for a single active X-chromosome in human somatic cells, but two active X-chromosomes in germ cells and in triploid cells with a 69,XXY karyotype. Studies of clonal populations of cells heterozygous for G6PD-A but expressing a single isozyme have provided evidence that the maintenance of the inactivation process is extremely stable, but that an occasional localized derepression event may occur. Such populations have also been used to show that methylation of X-chromosome DNA is not obviously different for XA and XI; furthermore, methylation is surprisingly unstable at least at some sites on the X. Studies of derepressed loci on the inactive X-chromosome in these clonal cell populations show that the expression of loci that escape inactivation is greater on XA than XI. G6PD variants have provided significant insights into the consequences of X-chromosome inactivation, revealing the role of intercellular communication and cell selection in determining female phenotype. The evidence that is now available indicates that the regulation of the X-chromosome is complex. The mechanisms involved include: 1) initiation, an event that may be mediated through an autosomal product and 2) maintenance which is stable but subject to programmed derepression of the entire chromosome during ontogeny of germ cells and occasional unprogrammed localized derepression in somatic cells. In addition, there may be transcriptional differences between XI and XA to compensate for monosomy of X-linked genes. The molecular basis for this multilevel regulation is unclear, but it seems certain that the cloned DNA probe for G6PD [Persico et al, 1981] will provide further insights.

DNA methylation stabilizes X chromosome inactivation in eutherians but not in marsupials: evidence for multistep maintenance of mammalian X dosage compensation.

In marsupials and eutherian mammals, X chromosome dosage compensation is achieved by inactivating one X chromosome in female cells; however, in marsupials, the inactive X chromosomes is always paternal, and some genes on the chromosome are partially expressed. To define the role of DNA methylation in maintenance of X chromosome inactivity, we examined loci for glucose-6-phosphate dehydrogenase and hypoxanthine phosphoribosyltransferase in a North American marsupial, the opossum Didelphis virginiana, by using genomic hybridization probes cloned from this species. We find that these marsupial genes are like their eutherian counterparts, with respect to sex differences in methylation of nuclease-insensitive (nonregulatory) chromatin. However, with respect to methylation of the nuclease-hypersensitive (regulatory) chromatin of the glucose-6-phosphate dehydrogenase locus, the opossum gene differs from those of eutherians, as the 5' cluster of CpG dinucleotides is hypomethylated in the paternal as well as the maternal gene. Despite hypomethylation of the 5' CpG cluster, the paternal allele, identified by an enzyme variant, is at best partially expressed; therefore, factors other than methylation are responsible for repression. In light of these results, it seems that the role of DNA methylation in eutherian X dosage compensation is to "lock in" the process initiated by such factors. Because of similarities between dosage compensation in marsupials and trophectoderm derivatives of eutherians, we propose that differences in timing of developmental events--rather than differences in the basic mechanisms of X inactivation--account for features of dosage compensation that differ among mammals.

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and deletion mapping: evolutionary significance.

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.

Clusters of CpG dinucleotides implicated by nuclease hypersensitivity as control elements of housekeeping genes.

DNA sequences of the X-chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) and glucose 6-phosphate dehydrogenase (G6PD) genes have revealed the presence of clusters of CpG dinucleotides, raising the possibility that such clusters are involved in the control of expression of these genes, which are expressed in all tissues. Although CpG clusters are not exclusive features of the X chromosome, the analysis of X-linked genes provides the means to determine whether CpG clusters are control elements; one of the two homologous X loci in female mammals is not expressed, so that active and inactive versions of the gene can be compared. In fact, it has been shown that these CpG clusters are undermethylated when the gene is active and extensively methylated when the gene is inactive. In addition to hypomethylation, chromatin hypersensitivity to endonuclease digestion is a known hallmark of regulatory sequences in eukaryotic genes. We report here that the CpG clusters of the active hprt and g6pd genes are not only undermethylated, but also hypersensitive to MspI, DNase I and S1 nuclease, further supporting the suggestion that they are involved in the control of expression of these genes.

In search of non-random X inactivation: studies of fetal membranes heterozygous for glucose-6-phosphate dehydrogenase.

Extraembryonic membranes and fetal tissues were obtained from 55 specimens of 5--11 weeks conceptual age. The glucose-6-phosphate dehydrogenase (G6PD) electrophoretic phenotype was determined and correlated with that of maternal blood. Fifteen specimens were heterozygous for G6PD A, and for nine of these the maternal allele could be determined. In none of these specimens did the isozyme pattern of the membraneous chorion or chorionic villi differ significantly from that of fetal tissue. We have obtained no evidence of non-random inactivation in extraembryonic membranes of human fetal specimens at this stage of development.