Search

Background Rigorous preclinical testing of soft tissue fillers has been lacking. No animal model has emerged as an accepted standard to evaluate tissue filler longevity. Objective To validate a small animal model to compare soft tissue filler degradation and tissue reaction. Methods Preliminary experiments compared caliper with magnetic resonance imaging volumetric analysis. Next, four hyaluronic acid (HA) fillers were injected into the dermis of Sprague–Dawley rats. The three dimensions of the implants were measured at day 0, day 1, and monthly for 1 year or complete resorption of the filler. Volumetric, histologic, and statistical analyses were performed. Results Magnetic resonance imaging results validated caliper-based volumetric measurements. Histology demonstrated injections in the subcutaneous space just deep to the dermis and panniculus carnosus. High- and very high-concentration HA fillers maintained significantly greater volumes and volume ratios than low-concentration HA fillers throughout the duration of the study. Conclusions The rat subcutis model demonstrated the ability to differentiate between HA fillers with different residence times. The caliper-based rat-subcutis method demonstrated consistent volumetric analysis and correlated with human residence times of HA fillers. These quantitative results validate the rat subcutis model as an expedited preclinical model for HA fillers.

OCT3/4 is a POU domain transcription factor that is critical for maintenance of pluripotency and self-renewal by embryonic stem (ES) cells and cells of the early mammalian embryo. It has been demonstrated to bind and regulate a number of genes, often in conjunction with the transcription factors SOX2 and NANOG. In an effort to further understand this regulatory network, chromatin immunoprecipitation was used to prepare a library of DNA segments specifically bound by OCT3/4 in undifferentiated mouse ES (mES) cell chromatin. One segment corresponds to a region within the first intron of the gene encoding histone deacetylase 4 (Hdac4), a Class II histone deacetylase. This region acts as a transcriptional repressor and contains at least two functional sites that are specifically bound by OCT3/4. HDAC4 is not expressed in the nuclei of OCT3/4+ mES cells and is upregulated upon differentiation. These findings demonstrate the participation of OCT3/4 in the repression of Hdac4 in ES cells.

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and beta-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.

X inactivation--the mammalian method of X chromosome dosage compensation--is extremely stable in human somatic cells; only fetal germ cells have a developmental program to reverse the process. The human placenta, at term, differs from other somatic tissues, since it has the ability to reverse the X-inactivation program. To determine whether reversal can be induced at other stages of placental development, we examined earlier placental specimens using a cell-hybridization assay. We found that global X reactivation is also inducible in villi cells from first-trimester spontaneous abortions but not from first-trimester elective terminations. These differences in inducibility are not associated with detectable variation in histone H4 acetylation, DNA methylation, or XIST expression--hallmarks of the inactivation process--so other factors must have a role. One notable feature is that the permissive cells, unlike nonpermissive ones, have ceased to proliferate in vivo and are either beginning or in the process of programmed cell death. Cessation of mitotic proliferation also characterizes oocytes at the stage at which they undergo X reactivation. We suggest that, along with undermethylation, the apoptotic changes accompanying cessation of cell proliferation contribute to the reversal of inactivation, not only in placental cells, but also in oocytes entering meiosis.

Human pluripotent stem cells would be invaluable forin vitrostudies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Publisher Summary Embryonic germ (EG) cells are pluripotent stem cells derived from primordial germ cells (PGCs) that arise in the late embryonic and early fetal period of development. Embryonic stem (ES) cells were first derived from the inner cell mass of mouse pre-implantation embryos, and EG cells were initially derived from mouse PGCs. Subsequently, EG cells have been derived from chicken, pig, and human PGCs. Pig, chicken, and mouse EG cells have been demonstrated to contribute to experimentally produced chimeric animals, including germline transmission in the latter two species. Human EG cells can be derived from PGCs by using methods similar to those used to derive mouse EG cultures. Like mouse embryonic stem and EG cells, human EG cells require leukemia inhibitory factor (LIF) for proliferation as undifferentiated stem cells. Unlike mouse EG cells, however, human EG cells do not readily lose their dependence on exogenous cytokines and factors supplied by the feeder layer, and they have a higher frequency of spontaneous differentiation into embryoid bodies (EBs). Although EBs are a loss to the pluripotent stem cell population, they are a source of cells expressing markers of mature cellular phenotypes, as well as their presumed progenitors and precursors. Cells that retain a high capacity for cell proliferation and express makers of multiple lineages can be isolated from EBs, and can be used in a variety of in vitro and in vivo differentiation paradigms. The current challenges are to match individual EB-derived (EBD) cultures to desired endpoints, and to enrich or purify populations of cells within EBD cultures to more specifically address biological requirements.

Soft tissue reconstruction often requires multiple surgical procedures that can result in scars and disfiguration. Facial soft tissue reconstruction represents a clinical challenge because even subtle deformities can severely affect an individual’s social and psychological function. We therefore developed a biosynthetic soft tissue replacement composed of poly(ethylene glycol) (PEG) and hyaluronic acid (HA) that can be injected and photocrosslinked in situ with transdermal light exposure. Modulating the ratio of synthetic to biological polymer allowed us to tune implant elasticity and volume persistence. In a small-animal model, implanted photocrosslinked PEG-HA showed a dose-dependent relationship between increasing PEG concentration and enhanced implant volume persistence. In direct comparison with commercial HA injections, the PEG-HA implants maintained significantly greater average volumes and heights. Reversibility of the implant volume was achieved with hyaluronidase injection. Pilot clinical testing in human patients confirmed the feasibility of the transdermal photocrosslinking approach for implantation in abdomen soft tissue, although an inflammatory response was observed surrounding some of the materials.

The severe phenotype of human females whose karyotype includes tiny ring X chromosomes has been attributed to the inability of the small ring X chromosome to inactivate. The XIST locus is expressed only from the inactive X chromosome, resides at the putative X inactivation center, and is considered a prime player in the initiation of mammalian X dosage compensation. Using PCR, Southern blot analysis, and in situ hybridization, we have looked for the presence of the XIST locus in tiny ring X chromosomes from eight females who have multiple congenital malformations and severe mental retardation. Our studies reveal heterogeneity within this group; some rings lack the XIST locus, while others have sequences homologous to probes for XIST. However, in the latter, the locus is either not expressed or negligibly expressed, based on reverse transcription-PCR analysis. Therefore, what these tiny ring chromosomes have in common is a level of XIST transcription comparable to an active X. As XIST transcription is an indicator of X chromosome inactivity, the absence of XIST transcription strongly suggests that tiny ring X chromosomes in females with severe phenotypes are mutants in the X chromosome inactivation pathway and that the inability of these rings to inactivate is responsible for the severe phenotypes.

The authors report studies of a female with severe hemophilia A resulting from a complex de novo translocation of chromosomes X and 17 (46,X,t(X; 17)). Somatic cell hybrids containing the normal X, the der(X), or the der(17) were analyzed for coagulation factor VIII (F8C) sequences using Southern blots and polymerase chain reaction. The normal X, always late replicating, contains a normal F8C gene, whereas the der(X) has no F8C sequences. The der(17) chromosome containing Xq24-Xq28 carries a functional G6PD locus and a deleted F8C allele that lacks exons 1--15. Also, it lacks the DXYS64-X locus, situated between the F8C locus and the Xq telomere. These results indicate that a cryptic breakpoint within Xq28 deleted the 5[prime] end of F8C, but left the more proximal G6PD locus intact on the der(17)chromosome. As the deleted segment includes the 5[prime] half of F8C as well as the subtelomeric DXYS64 locus, F8C must be oriented on the chromosome with its 5[prime] region closest to the telomere. Therefore, the order of these loci is Xcen-G6PD-3[prime]F8C-5[prime]F8C-DXYS64-Xqtel. The analysis of somatic cell hybrids has elucidated the true nature of the F8C mutation in the pro-band, revealing a more complex rearrangement (three chromosomes involved) than that expected frommore » cytogenetic analysis, chromosome painting, and Southern blots. A 900-kb segment within Xq28 has been translocated to another autosome. Hemophilia A in this heterozygous female is due to the decapitation of the F8C gene on the der(17) and inactivation of the intact allele on the normal X. 27 refs., 5 figs., 1 tab.« less

GNAS is a complex gene that through use of alternative first exons encodes signaling proteins Galpha(s) and XLalphas plus neurosecretory protein NESP55. Tissue-specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune-Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of Galpha(s), NESP55, and XLalphas prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of Galpha(s) was biallelic (maternal allele: 52.6%+/- 2.5%; paternal allele: 47.2%+/- 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%+/- 10%; paternal allele: 18.1%+/- 10%; p= 0.002) and XLalphas was preferentially expressed from the paternal allele (maternal allele: 2.7%+/- 0.3%; paternal allele: 97.3%+/- 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5-11 weeks postfertilization, and that imprinting of XLalphas occurs very early postfertilization. By contrast, imprinting of Galpha(s) most likely occurs after 11 weeks postfertilization and after tissue differentiation.

Human pluripotent stem cells (hPSCs) have been derived from the inner cell mass cells of blastocysts (embryonic stem cells) and primordial germ cells of the developing gonadal ridge (embryonic germ cells). Like their mouse counterparts, hPSCs can be maintained in culture in an undifferentiated state and, upon differentiation, generate a wide variety of cell types. Embryoid body (EB) formation is a requisite step in the process of in vitro differentiation of these stem cells and has been used to derive neurons and glia, vascular endothelium, hematopoietic cells, cardiomyocytes, and glucose-responsive insulin-producing cells from mouse PSCs. EBs generated from human embryonic germ cell cultures have also been found to contain a wide variety of cell types, including neural cells, vascular endothelium, muscle cells, and endodermal derivatives. Here, we report the isolation and culture of cells from human EBs as well as a characterization of their gene expression during growth in several different culture environments. These heterogeneous cell cultures are capable of robust and long-term [>70 population doublings (PD)] proliferation in culture, have normal karyotypes, and can be cryopreserved, clonally isolated, and stably transfected. Cell cultures and clonal lines retain a broad pattern of gene expression including simultaneous expression of markers normally associated with cells of neural, vascular/hematopoietic, muscle, and endoderm lineages. The growth and expression characteristics of these EB-derived cells suggest that they are relatively uncommitted precursor or progenitor cells. EB-derived cells may be suited to studies of human cell differentiation and may play a role in future transplantation therapies.

A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down’s syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event.

As a result of selection following random X chromosome inactivation in human females, X chromosomes with visible deletions are usually inactive in every somatic cell. We have studied a female with mental retardation and dysmorphic features whose karyotype includes an X chromosome with a visible interstitial deletion in the proximal long arm. Based on cytogenetic analysis, the proximal breakpoint appeared to be in band Xq13.1, and the distal one in band q21.3. However, molecular analyses show that less of the q13 band is missing than cytogenetic studies indicated, as the deletion includes only loci from the region Xq13.3 to Xq21.31. Unexpectedly, studies of chromosome replication show that the pattern of X inactivation is random. Whereas the deleted X chromosome is late replicating in some cells from all tissues studied, it is early replicating in the majority of blood lymphocytes and skin fibroblasts, and is the active X chromosome in many of the hybrids derived from skin fibroblasts. As this chromosome is able to inactivate, it must include those DNA sequences from the X-inactivation center (XIC) that are essential for cis X inactivation. Molecular studies show that the XIC region, at Xq13.2, is present, so it is unlikely that the lack of consistent inactivation of this chromosome is attributable to close proximity of the breakpoint to the XIC. Supporting this conclusion is the similarity of the breakpoints to those of the other chromosomes we studied, whose deletions clearly do not interfere with the ability to inactivate. Our results show that deletions distal to DXS441 in Xq13.2 do not interfere with cis X inactivation. We attribute the random pattern of X inactivation reported here to the fact that in the tissues studied, cells with this interstitial deletion are not at a selective disadvantage.

X chromosome dosage compensation in Marsupials is like that in eutherian mammals except that the paternal X chromosome is always inactive, and silence of this chromosome is not well maintained. We previously showed that the unstable inactivation of the paternal G6PD allele is associated with the lack of DNA methylation in the 5′ CpG cluster. Even though this CpG island is unmethylated, the paternal allele (marked by an enzyme variant) is at least partially and often severely repressed in most tissues of the opossum, so that factors other than methylation must inactivate the locus. Here we report that when cell cultures are established from these tissues, the silent G6PD locus is derepressed. Although often complete, the extent of derepression differs among tissues and within different cell types in the same tissue, and is not accompanied by obvious changes in the pattern of chromosome replication. Studies of the HPRT locus in these cells show that the paternal HPRT allele also derepresses in cultured cells. These observations suggest that without DNA methylation to maintain the silence of the locus, tissue or cell-specific factors act to repress the silent locus, but are unable to maintain inactivity through cell division, or are lost as cells proliferate in culture.

In mammals, X-chromosome dosage compensation is achieved by inactivating one X chromosome in female cells1. To test the hypothesis2 that genes on the silent X chromosome reactivate as a consequence of ageing, we examined the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 41 women who are heterozygous for mutations at this locus, leading to severe deficiency of the enzyme (Lesch-Nyhan syndrome3). We find that heterozygotes who are more than 10 yr old have an excess of HPRT+ skin fibroblast clones (59% rather than the 50% expected as a consequence of random X inactivation) but this excess does not increase with age. Further studies of eight of these heterozygotes show that the silent locus does not detectably reactivate spontaneously in culture, but only in response to treatment with 5-aza-2-deoxycytidine, a potent inhibitor of methylation. There is no age difference in the frequency of this reactivation as assayed by HATr clones, and a more sensitive autoradiographic assay shows only a twofold difference between young and old heterozy-gotes. Thus, age-related reactivation is not a feature of all X-linked loci, and may have species, tissue and locus-specific determinants.

To determine if expression of genes on the inactive X is inducible in human cells, we looked for reactivation events in a clone of fibroblasts transformed with origin-defective SV40. The karyotype of these cells was grossly heteroploid so that the aneuploidy associated with SV40 transformation occurs even in the absence of viral replication. This transformed clone, heterozygous for hypoxanthine phosphoribosyltransferase (HPRT), lacks HPRT activity, as the mutant allele is on the active X and the normal allele on the inactive X. Reactivation of the HPRT+ allele on the inactive X was observed at a frequency of 6 X 10(-5) per cell and increased approximately eightfold following treatment with the cytidine analogs 5-azacytidine (5azaC) and 5-azadeoxycytidine. The fact that spontaneous reactivation is detectable in some clones, but not all, suggests that the environment of the SV40-transformed cell, although not sufficient to induce generalized derepression, increases the frequency of rare reactivation events. The methylation pattern at the HPRT locus revealed transformation-associated alterations that may have predisposed these cells to reactivation events, spontaneous as well as 5azaC-induced.

We have established a cell line from mouse kidney cells expressing the tfm mutation and showed that these cells lack androgen binding activity. A subclone of these simian virus 40 (SV40)-transformed cells (6TGR-SV-tfm) selected in 6-thioguanine and lacking hypoxanthine phosphoribosyltransferase was used to produce a series of mouse--human hybrids containing the normal human X chromosome or various X autosome-translocation chromosomes (expressing only segments of the human X chromosome). When the androgen receptor locus (AR) was present in the hybrid, the number of receptor sites and kinetics of binding were similar to that in the human parental cells. Analysis of hybrids with partial human X chromosomes by using assays for X chromosome-linked enzymes and for the androgen receptor protein indicate that the AR locus on the human X chromosome is near the centromere between Xq13 and Xp11 and is proximal to the locus for phosphoglycerate kinase. Hybrids derived from 6TGR-SV-tfm mouse cells and human labial fibroblasts from an XY individual with the ar- form of androgen insensitivity have no binding activity. The lack of complementation indicates that the X chromosome-linked mutations in mouse and man affect homologous loci and supports the evolutionary conservation of X chromosomal loci in mammals; however, the position of the locus on the human X chromosome indicates that intrachromosomal rearrangement has occurred.

Mammalian sex-dosage compensation is mediated by maintaining activity of only one X chromosome. The asynchronous DNA synthesis characterizing the silent human X chromosome is thought to be reversible only during ontogeny of oocytes. We have previously shown that the glucose-6-phosphate dehydrogenase (G6PD) locus (G6PD) on the allocyclic X chromosome in chorionic villi is partially expressed. We now show that in hybrids derived from a clone of chorionic villi cells (heterozygous for G6PD A) and mouse A9 cells, the loci for G6PD, hypoxanthine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase are expressed on both human X chromosomes; the human X chromosomes carrying either G6PD A or B replicate synchronously with each other and with murine chromosomes. The X chromosome with G6PD A was identified as the original late-replicating X, because methylation in the body of the HPRT gene on this chromosome remained characteristic of the inactive X chromosome. These results indicate that X-chromosome inactivation is completely reversible in cells of trophoblast origin; induction of full transcriptional activity is accompanied by acquisition of isocyclic replication, showing an intimate relationship between these processes. The molecular events responsible for this reversal may be similar to those occurring during maturation of oocytes. Chorionic villi and derivative hybrids provide in vitro models for exploring early events that program the single active X chromosome.

The X chromosome in mammalian somatic cells is subject to unique regulation—usually genes on a single X chromosome are expressed while those on other X chromosomes are inactivated1. The X-locus for steroid sulphatase (STS; EC 3.1.6.2), the microsomal enzyme that catalyses the hydrolysis of various 3β-hydroxysteroid sulphates, is exceptional because it seems to escape inactivation. Evidence for this comes from fibroblast clones in females heterozygous for mutations that result in a severe deficiency of this enzyme in affected males; all clones from these heterozygotes have STS activity, and enzyme-deficient clones that are expected if the locus were subject to inactivation2, have not been found3. Further evidence that the STS locus escapes inactivation is that the human inactive X chromosome contributes STS activity to mouse–human hybrid cells4. On the basis of these hybrid studies5,6 the STS locus has been mapped to the distal half of the short arm (p22–pter) of the human X chromosome. Although the STS locus on both X chromosomes in human female cells is expressed, quantitative measurements of STS activity in males and females do not accurately reflect the sex differences in number of X chromosomes7–13 (Table 1). The ratio of mean values for normal females to that of normal males is greater than 1:1 but less than the ratio of 2:1 expected if STS loci on all X chromosomes were equally expressed. The incomplete dosage effect suggests that the STS locus on the inactive X chromosome might not be fully expressed. To test this hypothesis, we examined two heterozygotes for X-linked STS deficiency who were also heterozygous for the common electrophoretic variants of glucose-6-phosphate dehydrogenase (G6PD A and B). Studies of fibroblast clones from these females provide evidence, presented here, for differential expression of STS loci on the active and inactive X chromosome.

THE N-acetyl-β-D-glucosaminidase (hexosaminidase) activity in cultured human fibroblasts consists of at least three components (A, B and C)1–3. At least two of these (designated Hex A and Hex B) seem to be closely related, for (1) antiserum against Hex A reacts against Hex B, and vice versa4,5 (2) in Tay-Sachs disease only Hex A activity is deficient, accompanied by an increase in Hex B activity in certain tissues1,6, and (3) both Hex A and Hex B activities are missing in Sandhoff disease, another autosomal recessive disorder7. But in spite of several theories8–11, the precise relationship between these components remains unknown, as does the nature of the genetic and biochemical relationships between the two diseases. To investigate these questions we have fused Tay-Sachs with Sandhoff fibroblasts and obtained cultures containing heterokaryons which produce a hexosaminidase which is absent from the parent lines. It has the electrophoretic and heat lability characteristics of the Hex A found in normal fibroblasts.

Studies of glucose-6-phosphate dehydrogenase (G6PD) in heterozygous cells from chorionic villi of five fetal and one newborn placenta show that the locus on the allocyclic X is expressed in many cells of this trophectoderm derivative. Heterodimers were present in clonal populations of cells with normal diploid karyotype and a late replicating X chromosome. The expression of the two X chromosomes was unequal, based on ratios of homodimers and heterodimers in clones. Studies of DNA, digested with Hpa II and probed with cloned genomic G6PD sequences, indicate that expression of the locus in chorionic villi is associated with hypomethylation of 3' CpG clusters. These findings suggest that dosage compensation, at least at the G6PD locus, has not been well established or maintained (or both) in placental tissue. Furthermore, the active X chromosome in these human cells of trophoblastic origin can be either the paternal or maternal one; therefore, paternal X inactivation in extraembryonic lineages is not an essential feature of mammalian X dosage compensation.

Studies of a unique clone of skin fibroblasts from a normal 46 XX female reveal that the G6PD locus on the inactive X chromosome has been derepressed. The reactivation event occurs spontaneously, and is associated with normal karyotype, including the presence of a late-replicating X chromosome. Analysis of mouse-human hybrids with the relevant chromosome provides evidence that the derepressed locus is on the inactive X, and that reactivation is not extensive (the PGK locus is not derepressed). Nor is any general change in DNA methylation of this chromosome detectable with Hpa II and an X-specific DNA probe. Studies of the glucose-6-phosphate dehydrogenase phenotype in these heterozygous cells indicate that the reactivated X produces only half the enzyme subunits as are produced by the active X. Although this dosage difference may be related to the mutational event responsible for derepression of the locus, these observations along with other evidence suggest that loci on the inactive X, when expressed, have less activity than corresponding loci on the active X.

Skin fibroblasts of human males affected with adrenoleukodystrophy (ALD) have previously been shown to be abnormal with respect to C26 fatty acid content. Skin fibroblast clones from heterozygotes in three families segregating this mutation have been analyzed and are of two types: clones with normal ratios of C26 to C22 fatty acids and clones with an excess of C26 fatty acids similar to that found in cells of affected males. This indicates not only that the locus is X linked but also that it is subject to inactivation. In most of the heterozygotes there were significantly more clones of abnormal type than those expressing the normal allele, indicating a proliferative advantage in vitro for skin fibroblasts of mutant type. The increased levels of fatty acids in plasma in most heterozygotes and the phenotype of blood cells of women heterozygous for both ALD and glucose-6-phosphate dehydrogenase (G6PD) in one family are evidence that selection favoring the mutant allele may occur in vivo as well as in vitro and may explain why many heterozygotes manifest clinical symptoms of the disease. These studies have also revealed the close linkage between ALD and G6PD loci, because there are no recombinants among 18 informative offspring of doubly heterozygous mothers. Therefore, the ALD locus can be mapped on the human X chromosome near the G6PD locus at Xq28.

To explore the molecular basis of X chromosome inactivation, we have examined the human locus for glucose-6-phosphate dehydro-genase (G6PD) in various human tissues. Studies of DNA from males and females and from somatic cell hybrids with active or inactive X chromosomes, show that two remarkably dense clusters of CpG dinucleotides in the 3' coding sequences are hypomethylated in active G6PD genes but extensively methylated in inactive ones. Reacquisition of G6PD activity, either spontaneous or induced by 5-azacytidine, is accompanied by demethylation of both clusters; however, the clusters remain methylated in reactivants that express HPRT but not G6PD. Our observations implicate these 3' CpG clusters in the transcription of G6PD and in maintenance of dosage compensation for X linked housekeeping genes.