Biomedical subjects

V M Chapman

Publications and source records attributed to V M Chapman.

At least 109 records · Page 6Linked to original sources

PubMed1983

Differences in the DNA of the inactive X chromosomes of fetal and extraembryonic tissues of mice.

We have examined the role of DNA modification in X chromosome inactivation of fetal tissues of the mouse using DNA-mediated gene transfer for the gene hypoxanthine phosphoribosyltransferase (HPRT). Two types of tissues have been examined with respect to randomness of inactivation in 14-day mouse conceptuses: 1) fetal tissue, which undergoes random inactivation of either the maternal or paternal X; and 2) yolk sac endoderm tissue, an extraembryonic membrane, which normally undergoes nonrandom inactivation of the paternal X. Exploiting an electrophoretic variant of HPRT as a means to mark the active and inactive HPRT alleles we provide evidence that: 1) inactive X DNA of the fetus at 14 days behaves like that of both adult tissue and cell lines in that the inactive X DNA is not efficient in gene transfer; and 2) in contrast, inactive X DNA from yolk sac endoderm is functional in gene transfer. Thus, despite the similarity in single active X chromosome expression in yolk sac endoderm and somatic tissues, there appears to be a difference at the level of DNA modification between these two tissues.

Animals↗

PubMed1983

Trans-acting temporal locus within the beta-glucuronidase gene complex.

Mice carrying the [Gus]H haplotype of the beta-glucuronidase gene complex have considerably decreased enzyme levels and a decreased rate of enzyme synthesis. This is now shown to result from the action of two regulatory loci within the gene complex. One is a systemic regulator, Gus-u, that acts cis to cause a uniform reduction in enzyme levels in all tissues. The other is a temporal locus, Gus-t, that acts trans to cause abrupt switches in the rate of enzyme synthesis in only certain tissues and at characteristic stages of development. The distinction between these two loci was made possible by the introduction of a method for quantitating the relative numbers of A and H allozyme subunits in beta-glucuronidase tetramers. The procedure involves purification of the enzyme, cleavage at methionyl residues with CNBr, isoelectric focusing to separate the peptides, and quantitation of the peptide containing the A/H amino acid substitution. The presence of a trans-acting regulatory locus within a gene complex raises evolutionary and functional questions about why it is located there and how it acts.

Aging↗

PubMed1983

Electrophoretic variation for X chromosome-linked hypoxanthine phosphoribosyl transferase (HPRT) in wild-derived mice.

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.

Animals↗

PubMed1983

Somatic and germline mosaicism in interspecific chimaeras between Mus musculus and Mus caroli.

Detailed analysis of mosaicism in interspecific chimaeras between Mus musculus and Mus caroli revealed that cells of the two species could coexist and interact normally in all tissues studied. No selection occurred against M. caroli cells during gestation of chimaeras in the M. musculus uterus, but some tissue-specific differential growth of M. musculus and M. caroli cells occurred during postnatal development. Similar effects have, however, been reported in interstrain M. musculus chimaeras. The similarity between inter- and intraspecific chimaeric growth patterns supports the use of this interspecific system as a model for analysing cell lineage relationships during development.

Animals↗

PubMed1983

Use of repetitive DNA sequences to distinguish Mus musculus and Mus caroli cells by in situ hybridization.

Mammalian chimaeras have proved useful for investigating early steps in embryonic development. However, a complete clonal analysis of cell lineages has been limited by the lack of a marker which is ubiquitous and can distinguish parental cell types in situ. We have developed a cell marker system which fulfils these criteria. Chimaeric mice were successfully produced from two mouse species which possess sufficient genetic differences to allow unequivocal identification of parental cell types. DNA-DNA in situ hybridization with cloned, species-specific sequences was performed to distinguish the parental cell types. We have identified a cloned, Mus musculus satellite DNA sequence which shows hybridization differences between Mus musculus and Mus caroli DNA. This clone was used a a probe in in situ hybridizations to bone marrow chromosomes from Mus musculus, Mus caroli, and an interspecific F1 hybrid. The clone could qualitatively distinguish Mus musculus from Mus caroli chromosomes after in situ hybridization, even when they were derived from the same F1 hybrid cell. Quantitation of this hybridization to interphase nuclei from bone marrow spreads indicates that the probe can successfully distinguish Mus musculus from Mus caroli cells and can determine the percentage contribution of Mus musculus in mixtures of bone marrow cells of these species and in chimaeric bone marrow cell preparations.

Animals↗

PubMed1983

Identification of embryonic cell lineages in histological sections of M. musculus in-equilibrium M. caroli chimaeras.

An in situ cell marker system has been developed which allows identification of Mus caroli and Mus musculus cells in interspecific chimaeras. A radioactively labelled, cloned DNA probe to M. musculus satellite DNA was hybridized in situ to sections of M. musculus and M. caroli adult tissues. Autoradiography revealed high levels of hybridization to the nuclei of M. musculus cells, but little or no label bound to M. caroli cells. The DNA probe could also distinguish M. musculus and M. caroli cells in the same tissue section. Patches of labelled and unlabelled cells were clearly identified in sections of adult chimaeric tissues and also in the embryonic ectoderm of 6.5-day embryonic chimaeras. The ability to recognize M. musculus and M. caroli cells in sections of chimaeras should provide a powerful new tool in analyses of cell lineages in both embryonic and adult mouse chimaeras. The marker system has several advantages over other marker systems so far developed, the most important of which is its ubiquity. Since it is a nuclear marker, only cells without nuclei should be unsuited to its use. The potential of the marker system has been shown by its use in demonstrating directly for the first time the postimplantation derivatives of inner cell mass and trophectoderm in blastocysts 'reconstituted' with M. musculus trophectoderm and M. caroli inner cell mass.

Animals↗

PubMed1982

Evidence for DNA modification in the maintenance of X-chromosome inactivation of adult mouse tissues.

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 X-chromosome 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.

Animals↗

PubMed1982

Interspecific chimeras in mammals: a new experimental system.

The first viable interspecific chimeras in mammals have been made by mixing embryonic cells from two species of mouse, Mus musculus and Mus caroli. These chimeras resemble intraspecific chimeras in M. musculus in their patterns of mosaicism. They provide an interesting experimental system for studying interactions between cells of the two species. Three separate but related areas of investigation utilizing these chimeras are described. 1) In situ DNA-DNA hybridization using a cloned repetitive DNa sequence from M. musculus has enabled M. caroli and M. musculus cells to be distinguished in bone marrow spreads from chimeras. Further refinement of this technique should allow use of this in situ marker system for detailed analysis of cell distribution in chimeras. Such a ubiquitous marker system is not yet available in M. musculus. 2) Breeding interspecific chimeras has increased production of interspecific hybrids, which are very useful for studies of genetic interactions between the two species. 3) Mus caroli embryonic cells can survive in the M. musculus uterus in a chimera, although M. caroli embryos alone die around d 11 to 16 of pregnancy in M. musculus. Investigation of this phenomenon has implicated immune effector mechanisms in death of M. caroli embryos and has revealed the importance of trophoblast genotype for survival of foreign cells in the uterine environment.

Animals↗

PubMed1981

X chromosome reactivation in oocytes of Mus caroli.

Mature mammalian oocytes have both of their X chromosomes active, while somatic cells from the same individual have one of their X chromosomes in an inactive state. We asked whether the X chromosomes of the germ cells never undergo inactivation in their ontogeny or whether inactivation of an X chromosome does occur but is followed by a subsequent reactivation event. Our approach has used an electrophoretic polymorphism for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) in the mouse species Mus caroli. G6PD is dimeric, and a heterodimer is produced in cells from heterozygous females if and only if both X chromosomes are active. Ovaries from heterozygous fetuses at different gestational ages were dissected and either studied cytologically or pressed between microscopy slides to obtain germ cell-rich and germ cell-poor preparations. No heterodimer band was detected on the 10th day of development in germ cell-rich preparations. On subsequent days, an increasingly intense heterodimer band was detected, which, by the 13th day, was approximately twice as intense as the corresponding homodimer bands. Consideration of (i) the G6PD activity per germ cell and per somatic cell and (ii) the percentage of germ cells in the germ cell-rich preparations indicated that a heterodimer band should have been visible on the 10th day had both X chromosomes been active. Cytological examinations showed that the earliest germ cells enter meiotic prophase on the eleventh day. These results demonstrate that oogonia have a single active X chromosome and that the inactive X chromosome is reactivated at or, more likely, shortly before entry into meiotic prophase.

Alleles↗

PubMed1980

Quantitation of cis versus trans regulation of mouse beta-glucuronidase. Action of alleles of the Gus-r locus determining androgen inducibility.

Genetic studies have shown that the induction of beta-glucuronidase in mouse kidney in response to androgens is under the control of the Gus-r gene, closely linked to the beta-glucuronidase structural gene, Gus-s, on mouse Chromosome 5. Despite the fact that a single structural gene codes for beta-glucuronidase, enzyme molecules show considerable charge heterogeneity, presumably as a result of post-translational events. Because the enzyme is a tetramer, this heterogeneity has complicated examination of the cis versus trans action of the Gus-r gene. In order to resolve the problem, we have developed a quantitative method for determining the amount of beta-glucuronidase derived from each structural allele in mice heterozygous for beta-glucuronidase electrophoretic mobility. The method involves separation and quantitation of genetically variant cyanogen bromide peptides from the enzyme. Using this technique, we show that Gus-r acts cis, suggesting that it directly affects the template activity of the glucuronidase structural gene.

Alleles↗

PubMed1980

Intrinsic and extrinsic factors affecting the viability of Mus caroli x M. musculus hybrid embryos.

Interspecific hybrids between M. musculus and M. caroli, a wild species of mouse, were produced by artificial insemination, although the species do not normally interbreed. However, the success rate was low, with many embryos dying at various stages of pregnancy. Hybrid embryos were retarded in comparison with either parent species from the earliest stages of development, suggesting that intrinsic problems of genomic incompatibility play a major role in poor hybrid survival. However, failure of normal embryo-uterine interactions may also be important, since M. caroli x M. caroli embryos transferred to the M. musculus uterus also failed to survive to term. It is suggested that a maternal immune response to antigens on the foreign trophoblast may be involved.

Animals↗

PubMed1980

Expression of the maternally derived X chromosome in the mural trophoblast of the mouse.

Only the maternally derived allelic form of the X-chromosome-linked enzme phosphoglycerate kinase (PGK-1) is observed in the mural trophoblast of heterozygous female progeny in F1 and backcross matings. We have demonstrated that this expression of the maternally derived PGK-1 is not a result of maternal tissue contamination nor of selection of cells expressing the maternal X chromosome (Xm). Our results suggest that the expression of Xm in mural trophoblast is a consequence of nonrandom X-chromosome inactivation in trophectoderm cells.

Animals↗

PubMed1979

Paternal X chromosome expression in extraembryonic membranes of XO mice.

The paternally derived allelic form of the X chromosome linked enzyme phosphoglycerate kinase (PGK-1) is expressed in both the fetus and extraembryonic membranes of 9.5 day post coitum XO (XpO) mouse conceptuses. Previous studies on trophectoderm and primitive endoderm derived extraembryonic membranes in XX conceptuses suggest expression of only the maternally derived X chromosome (Xm). Our results suggest that the observed lack of expression of Xp in the trophectoderm and primitive endoderm derivatives of XX embryos is not an intrinsic property of the Xp chromosome.

Alleles↗

PubMed1978