Barbara R. Migeon

Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome.

We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres.

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 3H-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 3H-guanine, is evidence that the locus for hypoxanthineguanine phosphoribosyl transferase on one of the X chromosomes is inactive.

Sex difference in methylation of single-copy genes in human meiotic germ cells: Implications for X chromosome inactivation, parental imprinting, and origin of CpG mutations

To determine the methylation status of female germ cells in reference to the programmed reversal of X chromosome inactivation in these cells, we examined human fetal ovaries at developmental stages from the time germ cells initiate meiosis to when they cease to synthesize DNA (8–21 weeks gestation). Using methylation-sensitive restriction enzymes, we analyzed 57 MspI sites (32 sites in the CpG islands, and 25 nonclustered sites) from five X-linked housekeeping genes (HPRT, G6PD, P3, PGK, and GLA) and two tissue specific genes (X-linked F9 and autosomal EPO). Methylation patterns were compared to those of male germ cells, sperm, and somatic tissues of both sexes. All 32 MspI sites in CpG islands were unmethylated in germ-cell fractions of fetal ovary and adult testes, which could explain the reversibility of X inactivation in these tissues. However, whereas male meiotic germ cells were extensively methylated outside the islands (in the body of genes) and the methylation patterns resembled those of most somatic tissues, none of the 25 nonclustered CpGs was methylated in DNA contributed by the germ-cell component of fetal ovaries. The presence of faint MspI-like fragments in HpaII digests of fetal testes as well as fetal ovary prior to the onset of meiosis suggests that DNA of primordial germ cells is unmethylated in both sexes. Our observations of meiotic germ cells suggest that the female germ cells remain unmethylated, but that methylation in male germ cells occurs postnatally, prior to or during the early stages of spermatogenesis. In any event, the striking sex difference in methylation status of endogenous single-copy genes in meiotic germ cells could provide a molecular basis for parental imprinting of the mammalian genome.

Complex chromosome rearrangements:Report of a new case and literature review

A complex and unique, apparently balanced translocation involving three autosomes and an X in a phenotypically abnormal child is described. Family studies using glucose 6 phosphate dehydrogenase as a marker provided biochemical evidence of non‐random expression of this Xq locus and suggested that this de novo abnormality in the proband could be paternal in origin ‐ the first such instance to be recorded.

Identification of TSIX, encoding an RNA antisense to human XIST, reveals differences from its murine counterpart: implications for X inactivation.

X inactivation is the mammalian method for X-chromosome dosage compensation, but some features of this developmental process vary among mammals. Such species variations provide insights into the essential components of the pathway. Tsix encodes a transcript antisense to the murine Xist transcript and is expressed in the mouse embryo only during the initial stages of X inactivation; it has been shown to play a role in imprinted X inactivation in the mouse placenta. We have identified its counterpart within the human X inactivation center (XIC). Human TSIX produces a >30-kb transcript that is expressed only in cells of fetal origin; it is expressed from human XIC transgenes in mouse embryonic stem cells and from human embryoid-body-derived cells, but not from human adult somatic cells. Differences in the structure of human and murine genes indicate that human TSIX was truncated during evolution. These differences could explain the fact that X inactivation is not imprinted in human placenta, and they raise questions about the role of TSIX in random X inactivation.

Human-Mouse Somatic Cell Hybrids with Single Human Chromosome (Group E): Link with Thymidine Kinase Actvity

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 3H-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.

Species Differences in TSIX/Tsix Reveal the Roles of These Genes in X-Chromosome Inactivation

Transcriptional silencing of the human inactive X chromosome is induced by the XIST gene within the human X-inactivation center. The XIST allele must be turned off on one X chromosome to maintain its activity in cells of both sexes. In the mouse placenta, where X inactivation is imprinted (the paternal X chromosome is always inactive), the maternal Xist allele is repressed by a cis-acting antisense transcript, encoded by the Tsix gene. However, it remains to be seen whether this antisense transcript protects the future active X chromosome during random inactivation in the embryo proper. We recently identified the human TSIX gene and showed that it lacks key regulatory elements needed for the imprinting function of murine Tsix. Now, using RNA FISH for cellular localization of transcripts in human fetal cells, we show that human TSIX antisense transcripts are unable to repress XIST. In fact, TSIX is transcribed only from the inactive X chromosome and is coexpressed with XIST. Also, TSIX is not maternally imprinted in placental tissues, and its transcription persists in placental and fetal tissues, throughout embryogenesis. Therefore, the repression of Xist by mouse Tsix has no counterpart in humans, and TSIX is not the gene that protects the active X chromosome from random inactivation. Because human TSIX cannot imprint X inactivation in the placenta, it serves as a mutant for mouse Tsix, providing insights into features responsible for antisense activity in imprinted X inactivation.

Genetic Inactivation of the α-Galactosidase Locus in Carriers of Fabry's Disease

Skin fibroblasts from a patient with Fabrys disease showed deficient activity of α-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 α-galactosidase locus and makes possible the detection of carriers of Fabrys disease even when the enzymatic activity in their leukocytes and uncloned fibroblasts is within the range of controls.

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.

Hybridization of somatic cells derived from mouse and Syrian hamster: Evolution of karyotype and enzyme studies

Somatic hybrids of drug-resistant mutant hamster and mouse cell lines have been isolated and propagated in long-term culture and have been studied in respect to karyotype and three enzymes. During the course of propagation the long-surviving hybrid clones show progressive loss of telocentric chromosomes associated in at least one case with loss of mouse enzyme. Hybrid clones showed hybrid molecules for malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6PGD) made up by recombination of parental subunits.