Biomedical subjects
S Rastan
Publications and source records attributed to S Rastan.
Genetic mapping in the region of the mouse X-inactivation center.
The mouse X-inactivation center lies just distal to the T16H breakpoint. Utilizing pedigree analysis of backcross progeny from a Mus domesticus/Mus spretus interspecific cross, we have mapped a number of genetic loci, gene probes, microclones, and EagI linking clones distal to the T16H breakpoint. The genetic analysis provides a detailed genetic map in the vicinity of the mouse X-inactivation center. Comparative mapping data from the human X chromosome indicate that the most probable location of the mouse X-inactivation center is distal to Ccg-1 and in the region of the Pgk-1 locus. We report the assignment of two new loci, EM13 and DXSmh44, to the Ccg-1/Pgk-1 interval.
Construction and analysis of linking libraries from the mouse X chromosome.
A hybrid cell line containing the mouse X chromosome on a human background has been used to construct linking libraries from the mouse X chromosome, and approximately 250 unique EagI and NotI clones have been identified. Seventy-three clones have been sublocalized onto the X chromosome using interspecific Mus spretus/Mus domesticus crosses and a panel of somatic cell hybrids carrying one-half of reciprocal X-autosome translocations. The average spacing of the linking clones mapped to date is about one every 2 Mb of DNA. Two clones from the central region of the chromosome have been physically linked by pulsed-field gel electrophoresis. A large number of clones contain conserved sequences, indicating the presence of CpG-rich island-associated genes. The clones isolated from these libraries provide a valuable resource for comparative mapping between man and mouse X chromosomes, isolation of X-linked disease loci of interest by reverse genetics, and analysis of the long-range structure and organization of the chromosome.
Age-related reactivation of an X-linked gene close to the inactivation centre in the mouse.
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Expression of H-2 class I genes in murine extra-embryonic tissues.
Murine major histocompatibility complex class I genes are transcribed at high levels in placental tissues, lower levels in yolk-sac tissues and at barely detectable levels in the embryo at Day 13.5 of gestation. Genes are expressed at equivalent levels whether inherited maternally or paternally, and the genetic background has no effect on class I gene transcription. These results show that potential alloantigens are expressed in extra-embryonic tissues intimately associated with maternal tissues and blood supply and yet fail to induce immunological rejection.
T-cell depletion of allogeneic bone marrow prevents acceleration of graft-versus-host disease induced by exogenous interleukin 2.
Highly purified human recombinant interleukin 2 (IL-2) markedly accelerated lethal GVHD in the H-2-identical B10.BR----CBA combination, but had no effect when the donor cells were depleted of mature (Thy-1.2-positive) T lymphocytes, indicating a strong immunopotentiating effect of IL-2 on mature T cells causing GVHD. In the same donor-host combination, IL-2 did not influence the recovery from the post-transplantation bone marrow aplasia. The results suggest that IL-2 could be considered for adjuvant hormonal therapy to enhance immune recovery in recipients of T-cell-depleted allogeneic marrow.
Saccharide structures of the mouse embryo during the first eight days of development. Inferences from immunocytochemical studies using monoclonal antibodies in conjunction with glycosidases.
Monoclonal anti-carbohydrate antibodies have been used in conjunction with glycosidases in immunofluorescence studies to derive information about the structures and in situ distribution of saccharides of the mouse embryo during the first 8 days of development. The salient findings are as follows: Branched poly-N-acetyllactosamine sequences of I-antigen type are detectable from the first day onwards and are widely distributed in cells of the endoderm, ectoderm and mesoderm. Linear poly-N-acetyllactosamine sequences of i-antigen type are detectable from the fifth day onwards in cells of all three lineages, but have a more restricted distribution than the sequences of I-type. Poly-N-acetyllactosamine sequences that are susceptible to digestion with endo-beta-galactosidase are the main carriers of the SSEA-1, C14 and the blood group B-like antigens, which have the following structures (Formula; see text) and are found in endoderm and ectoderm but not in mesoderm cells. In the trophoblast however, these antigens are borne on saccharides that are resistant to endo-beta-galactosidase. A proportion of the poly-N-acetyllactosamine structures in the endoderm and the ectoderm of the 5- and 6-day embryos may contain the following novel structures: (Formula; see text) in which antigenicities of SSEA-1 and C14 determinants are masked. There are several types of sialyl-oligosaccharides: those reactive with anti-Gd, which has a specificity for NeuAc alpha 2-3Gal beta 1-4GlcNAc sequence in the extraembryonic mesoderm and the heart; those reactive with anti-Pr2 but not with anti-Gd, which may correspond to other N-acetylneuraminic acid containing sequences such as NeuAc alpha 2-3Gal beta 1-3GalNAc or NeuAc alpha 2-6Gal in preimplantation embryos and in the yolk sac, neural ectoderm and mesenchyme of the 8-day embryo; those with other sialic acid forms or linkages that do not react with anti-Gd and Pr2; among these are sialosyl-i sequences in the extraembryonic ectoderm, and sialosyl-I sequences in most cell types during the first 8 days. The latter are the main poly-N-acetyllactosamine structures in the neural ectoderm of the 8-day embryo. The sequence Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc/GlcNAc, or cross-reactive structures, which bind FC10.2 antibody occur in the extraembryonic endoderm and yolk sac. The roles of specific carbohydrate structures as receptors during embryonic development and cell growth are important topics of current research.(ABSTRACT TRUNCATED AT 400 WORDS)
X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation.
The predictions of a model for the initiation of X-chromosome inactivation based on a single inactivation centre were tested in a cytogenetic study using six different embryo-derived (EK) stem cell lines, each with a different-sized deletion of the distal part of one of the X-chromosomes. Metaphase chromosomes were prepared by the Kanda method from each cell line in the undifferentiated state and after induction of differentiation, and cytogenetic evidence sought for a dark-staining inactive X-chromosome. The results confirm the predictions of the model in that when the inactivation centre is deleted from one of the X-chromosomes neither X present in a diploid cell can be inactivated, and in addition considerably further localize the position of the inactivation centre on the X-chromosome.
Cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly-N-acetyllactosamine series.
Roles of cell surface carbohydrates containing the 3-fucosyl-N-acetyllactosamine and poly-N-acetyllactosamine sequences (SSEA-1 and I antigens, respectively) in the compaction of mouse embryos have been investigated using the endo-beta-galactosidase of Bacteroides fragilis to modify the surface of cleavage-stage embryos. Treatment with this enzyme abolished SSEA-1 activity and diminished I antigen activity on the embryonic cell surface. Embryos cultured in the presence of endo-beta-galactosidase from the 2- to 4-cell stage onwards, or treated with the enzyme at the compacting 8-cell stage, continued to compact and proceeded to form blastocysts at the normal rate. However, when compacted 8- to 16-cell embryos were experimentally decompacted in calcium-free medium, treated for 1 h with endo-beta-galactosidase and returned to normal culture medium, the time taken for 50% of the embryos to recompact was prolonged five-fold. There was an even greater delay if these embryos were maintained in culture medium containing the enzyme. Blastocysts were eventually formed under both conditions. Thus, endo-beta-galactosidase did not affect compaction unless the embryos were first decompacted. On the assumption that recompaction and de novo compaction occur by similar mechanisms, we propose that carbohydrate-binding molecules are involved which have high affinities for poly-N-acetyllactosamine structures and protect them from digestion by endo-beta-galactosidase.
Parental source of chromosome imprinting and its relevance for X chromosome inactivation.
In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara. It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara. Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.
Non-random X-chromosome inactivation in mouse X-autosome translocation embryos--location of the inactivation centre.
X-chromosome inactivation was investigated cytologically using the modified Kanda method which differentially stains inactive X-chromosome material at metaphase in balanced 13 1/2-day female embryos heterozygous for four X-autosome rearrangements, reciprocal translocations T(X;4)37H, T(X;11)38H and T(X;16)16H (Searle's translocation) and the insertion translocation Is(7;X)1 Ct (Cattanach's translocation). In all cases non-random inactivation was found. In the reciprocal translocation heterozygotes only one translocation product ever showed Kanda staining. In addition in a proportion of cells from T(X;4)37H, T(X;11)38H and Is(7;X)1Ct the Kanda staining revealed differential staining of X-chromosome material and attached autosomal material within the translocation product. In a study of 8 1/2-day female embryos doubly heterozygous for Searle's translocation and Cattanach's translocation two unbalanced types of embryo were found. In one type of unbalanced female embryo of the karyotype 40(X(7)/X16;16/16) no inactivated X-chromosomal material is found. A second unbalanced type of female embryo, of the presumptive karyotype 40(X(7)/XN;16X/16) was found in which two inactivated chromosomes were present in the majority of metaphase spreads. A simple model for the initiation of X-chromosome inactivation based on the presence of a single inactivation centre distal to the breakpoint in Searle's translocation explains these findings.
Primary non-random X-inactivation caused by controlling elements in the mouse demonstrated at the cellular level.
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Timing of X-chromosome inactivation in postimplantation mouse embryos.
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X-chromosome inactivation in extra-embryonic membranes of diploid parthenogenetic mouse embryos demonstrated by differential staining.
In somatic cells of female mammals one of the two X chromosomes is genetically inactive and heterochromatic, resulting in dosage compensation for X-linked genes. In marsupials the paternally derived X chromosome is preferentially inactivated. In eutherian mammals, although either X chromosome can be inactivated at random in somatic cells, preferential inactivation of the paternally derived X chromosome has been demonstrated cytologically in mouse and rat yolk sac and mouse chorion and biochemically in mouse yolk sac, chorionic ectoderm and trophoblast. In mouse yolk sac the non-random element has been shown both biochemically and cytologically to be confined to the endoderm layer in which there is almost total paternal X-chromosome activity in the separated yolk sac layers of diploid parthenogenetic mouse embryos in which both X chromosomes are maternally derived. Kaufman et al. have demonstrated X inactivation in somatic cells of diploid parthenogenetic embryos, and we have used a modification of Kanda's method, which renders the presumptive inactive X dark staining, to reveal an inactive X chromosome in both endoderm and mesoderm layers of separated yolk sacs from parthenogenones. Thus even in tissues in which there is normally total non-random paternal X inactivation, in the absence of a paternally derived X chromosome a maternally derived X can be inactivated.
NIEHS/EPA Workshops. Genomic imprinting.
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The search for the mouse X-chromosome inactivation centre.
The phenomenon of X-chromosome inactivation in female mammals, whereby one of the two X chromosome present in each cell of the female embryo is inactivated early in development, was first described by Mary Lyon in 1961. Nearly 30 years later, the mechanism of X-chromosome inactivation remains unknown. Strong evidence has accumulated over the years, however, for the involvement of a major switch or inactivation centre on the mouse X chromosome. Identification of the inactivation centre at the molecular level would be an important step in understanding the mechanism of X-inactivation. In this paper we review the evidence for the existence and location of the X-inactivation centre on the mouse X-chromosome, present data on the molecular genetic mapping of this region, and describe ongoing strategies we are using to attempt to identify the inactivation centre at the molecular level.
Interaction between the Xce locus and imprinting of the paternal X chromosome in mouse yolk-sac endoderm.
In female eutherian mammals preferential inactivation of the paternally derived X chromosome (XP) takes place in certain extra-embryonic tissues such as mouse yolk-sac endoderm, chorionic ectoderm and trophoblast and has been demonstrated both biochemically and cytologically. This is thought to be due to the paternal X chromosome being 'imprinted', that is, somehow marked as different, during either male gametogenesis or fertilization, causing primary nonrandom X-inactivation in tissues that differentiate early, such as trophectoderm and primitive endoderm, from which yolk-sac endoderm is derived. Different alleles of the X-chromosome controlling element, Xce locus, centrally located on the mouse X chromosome, also cause primary nonrandom X-chromosome inactivation in embryonic tissues which would otherwise show random inactivation. The work reported here was designed to elucidate whether the nonrandom inactivation of the imprinted XP in yolk-sac endoderm could be modified, or even overridden, by the effect of different Xce alleles. Using the modified Kanda method we have therefore studied the proportion of cells at metaphase with the XP inactive in separated yolk-sac endoderm and mesoderm of mouse embryos heterozygous for a marker X chromosome (Cattanach's translocation) carrying different Xce alleles on XP and XM. The results show that the extreme Xcec allele, when present on the paternally derived X, can significantly reduce the proportion of inactive XP seen in yolk-sac endoderm compared with controls. This is the first evidence that imprinting of XP is not an 'all or none' event but can be modified by a 'strong' allele at the Xce locus, and is another indication that the Xce locus may represent the inactivation centre.