Relationship between anatomical structure and metabolism of plant tissues. I. Differences between the qualitative and quantitative composition of phenolic substances of apple explants and that of callus and cells produced by the culture]

Relationship between anatomical structure and metabolism of plant tissues. I. Differences between the qualitative and quantitative composition of phenolic substances of apple-fruit explants and that of calli and cells cultured from these explants. Phenolic compounds of intact apple-fruits (CV. Golden delicious), fruit fragments cultured on agar medium, newly formed calli and cell suspensions prepared from these calli, were studied quantitatively and qualitatively. The phenol content of the tissues decreased during the first days of culture, then recovered practically the initial level just before the initiating calli became visible. This content is very low in the calli and in the cultured cells. But the most remarkable result is that the qualitative compositions of the phenols extracted from the fruit tissues, the calli and the cells were different : p-coumaryglucose, which is abundant in the fruit, disappeared almost completely in the calli, in which three compounds were formed de novo, X1 which was tentatively identified as ferulylquinic acid, X2, a glycoside of p-coumaric acid different from p-coumarylglucose, and X3 not yet identified; the cells synthesized also X1 and X3 but not X2, and contained no p-coumaryglucose while feruylquinic acid was abundant. The study on the processes of induction or regulation of the enzymes implied in these metabolic modifications is under way.     

Imprint switching for non-random X-chromosome inactivation during mouse oocyte growth

In mammals, X-chromosome inactivation occurs in all female cells, leaving only a single active X chromosome. This serves to equalise the dosage of X-linked genes in male and female cells. In the mouse, the paternally derived X chromosome (X(P)) is imprinted and preferentially inactivated in the extraembryonic tissues whereas in the embryonic tissues inactivation is random. To investigate how X(P) is chosen as an inactivated X chromosome in the extraembryonic cells, we have produced experimental embryos by serial nuclear transplantation from non-growing (ng) oocytes and fully grown (fg) oocytes, in which the X chromosomes are marked with (1) an X-linked lacZ reporter gene to assay X-chromosome activity, or (2) the Rb(X.9)6H translocation as a cytogenetic marker for studying replication timing. In the extraembryonic tissues of these ng/fg embryos, the maternal X chromosome (X(M)) derived from the ng oocyte was preferentially inactivated whereas that from the fg oocyte remained active. However, in the embryonic tissues, X inactivation was random. This suggests that (1) a maternal imprint is set on the X(M) during oocyte growth, (2) the maternal imprint serves to render the X(M) resistant to inactivation in the extraembryonic tissues and (3) the X(M) derived from an ng oocyte resembles a normal X(P).     

Sex chromosome elimination,X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae)

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.     

An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta

A GFP transgene has been integrated on the proximal part of the mouse X chromosome just distal of Timp and Syn1. During development, this X-linked GFP transgene exhibits widespread green fluorescence throughout the embryonic and adult life of male mice but displays mosaic expression in tissues as a result of X-inactivation in females. In living female embryos, inactivation of the transgene is imprinted in extraembryonic regions and random in the embryo proper, demonstrating that this reporter is behaving in a similar fashion to the majority of X-linked loci, and so provides a vital readout of X chromosome activity. This is observation is further supported in T16H/X female mice harboring the GFP transgene on the normal X chromosome where reporter inactivation is observed in somatic cells. The differential expression of GFP activity facilitates fluorescence activated cell sorting for the purification of GFP+ vs. GFP- cells from female embryonic tissues, thereby allowing access to populations of cells that have kept active a particular X chromosome. By tracking the activity of this X-linked GFP transgene, we discovered that the primary and secondary giant cells of the X/X placenta maintain an active paternal copy of this transgene on the presumed silenced paternal X-chromosome. This finding implies that the imprint on the paternal X chromosome may be relaxed in these trophectodermal derivatives.     

X chromosome inactivation mosaicism in the mouse

A cytologically detectable mosaicism resulting from X-chromosome inactivation occurring in mice heterozygous for Cattanach's translocation has been used to examine the time of X chromosome inactivation, and the sizes of primordial precursor pools for lung, thymus, spleen, fascia, and melanocytes. The extent of covariance in mosaic composition among tissues within individuals suggests that, if X inactivation occurs randomly, it must occur after determination of embryoblast cells, at some time immediately before or after implantation, and that it must occur before divergence of mesoderm from ectoderm. The extent of independent variance among the various tissues is such as to suggest that none of them arise from primordial precursor pools smaller than 20 to 30 cells.     

Improved technique for the expression of fragile-X in cultured amniotic fluid cells

Summary An improved technique for inducing fra(X) expression in cultured cells was obtained by using diazepam for mitotic arrest and 5-fluorodeoxyuridine (FUdR) for the induction of fra(X) expression. The method was developed using cultured fibroblast and urinary cells from fra(X) patients. Prenatal studies were performed on cultured amniotic fluid cells in five pregnancies at risk for fra(X). In two cases the cultured cells showed a 46,XY, fra(X) karyotype. One of the pregnancies was terminated and the diagnosis was confirmed by chromosome studies on several fetal tissues including chorionic villi and by histopathologic changes in the lymphatic vessels of the fetal testes. The fra(X) was also demonstrated in chorionic villi in a case in which amniotic fluid cells were not studied. Chorionic villi were isolated after a spontaneous abortion, the cultured cells had a 45,X karyotype and in addition 5% of the cells were fra(X) positive.     

Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label

We used fracture-label to establish ultrastructural localization of glycoproteins in cross-fractured nuclei of duodenal columnar and exocrine pancreatic cells. Mannose residues were detected in cell nuclei by labeling freeze-fractured tissues with concanavalin A-horseradish peroxidase X colloidal gold (Con A-HRP X CG) or direct concanavalin A X colloidal gold (Con A X CG); fucose residues were detected with Ulex Europaeus I X colloidal gold (UEA I X CG) markers. Areas of the three main intranuclear compartments (euchromatin, heterochromatin, and nucleolus) exposed by freeze-fracture were determined by automated image analysis. Colloidal gold particles bound to each nuclear subcompartment were counted and the results expressed in number of colloidal gold particles per square micrometer +/- SEM. Duodenal and pancreatic tissues fractured and labeled with Con A-HRP X CG complex or direct Con A X CG conjugates showed that the vast majority of Con A binding sites was confined to euchromatin regions with only sparse labeling of the heterochromatin and nucleolus. UEA I labeling of duodenal columnar cells showed that colloidal gold particles were almost exclusively confined to cross-fractured areas where euchromatin is exposed. Trypsinization of the fractured tissues before labeling with Con A and UEA I abolished 95-100% of the original label. Our results show that, within the nucleoplasm, mannose and fucose are residues of glycoproteins preferentially located within the regions of euchromatin.     

Differential methylation of the hypervariable locus DXS255 on active and inactive X chromosomes correlates with the expression of a human X-linked gene

Consistent differences in methylation of particular cytosine residues in the DNA of active and inactive X chromosomes can be used for rapid, direct analysis of X-inactivation patterns in different female tissues. We have studied methylation of the highly polymorphic DXS255 locus in tissues from patients with deficiency of the E1 alpha subunit of the pyruvate dehydrogenase complex in whom the results can be correlated directly with total enzyme activity, levels of immunoreactive protein, and patterns of cell mosaicism. The results confirm that methylation of the DXS255 locus correlates with X-chromosome expression. In patients and normal controls, the pattern of X inactivation varied widely from tissue to tissue and often deviated markedly from a 50:50 proportion. These deviations are likely to reflect small numbers of tissue-specific stem cells at the time of random X inactivation and cannot be taken alone as evidence for selection or "nonrandom" inactivation.     

X chromosome inactivation in X autosome translocation carrier cows.

The pattern of X chromosome inactivation in X autosome translocation carries in a herd of Limousin-Jersey crossbred cattle was studied using the reverse banding technique consisting of 5-bromodeoxyuridine incorporation and acridine orange staining and autoradiography on cultures of solid tissues and blood samples exposed to tritiated thymidine. The late-replicating X chromosome was noted to be the normal X in strikingly high proportions of cells in cultures of different tissues from all translocation carriers. It is suggested that the predominance of cells in which the normal X is inactivated may be the result of a post-inactivation selection process. Such a selection process during the prenatal life favouring cells in which the genes of the normal X chromosome remain unexpressed in translocation carrier females may be the mechanism that helps these conceptuses escape the adverse effects of functional aneuploidy. Based on the observation that the translocation carriers of this line of cattle are exclusively females and that there is a higher than expected rate of pregnancy loss, it is also postulated that the altered X chromosome may be lethal to all male conceptuses and to some of their female counterparts.     

DNA methylation patterns in the 5S DNAs of Xenopus laevis

The frequency of cytosine methylation at specific sites in the somatic 5S DNA (X1s) and trace oocyte 5S DNA (X1t) of X. laevis has been determined using restriction enzymes that are inhibited by the presence of 5-methylcytosine (5mC) within their cleavage sequences. 5S DNA methylation patterns were determined in genomic DNA from mature red blood cells, which express neither type of 5S gene, and from liver, which expresses only X1s. All the sites examined in X1t are greater than 95% methylated in red cells and liver. In the X1s of red cells all the sites examined are methylated in greater than 95% of repeats, while in liver some sites are modified in only 90% of repeats. Repeats containing unmethylated sites are randomly distributed throughout the tandem arrays in both red cells and liver. The high levels of methylation for X1s are in marked contrast to the situation with other Xenopus genes which do have sites of significant undermethylation in tissues where they are active. Thus, undermethylation in active genetic regions may not be a general feature for all classes of eukaryotic genes.     

Instability of the CGG repeat and expression of the FMR1 protein in a male fragile X patient with a lung tumor.

The molecular mechanism of the fragile X syndrome is based on the expansion of an CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. This repeat displays instability both between individuals and within an individual. We studied the instability of the CGG repeat and the expression of the FMR1 protein (FMRP) in several different tissues derived from a male fragile X patient. Using Southern blot analysis, only a full mutation is detected in 9 of the 11 tissues tested. The lung tumor contains a methylated premutation of 160 repeats, whereas in the testis, besides the full mutation, a premutation of 60 CGG repeats is detected. Immunohistochemistry of the testis revealed expression of FMR1 in the spermatogonia only, confirming the previous finding that, in the sperm cells of fragile X patients with a full mutation in their blood cells, only a premutation is present. Immunohistochemistry of brain and lung tissue revealed that 1% of the cells are expressing the FMRP. PCR analysis demonstrated the presence of a premutation of 160 repeats in these FMR1-expressing cells. This indicates that the tumor was derived from a lung cell containing a premutation. Remarkably, despite the methylation of the EagI and BssHII sites, FMRP expression is detected in the tumor. Methylation of both restriction sites has thus far resulted in a 100% correlation with the lack of FMR1 expression, but the results found in the tumor suggest that the CpGs in these restriction sites are not essential for regulation of FMR1 expression. This indicates a need for a more accurate study of the exact promoter of FMR1.     

Involvement of headless myosin X in the motility of immortalized gonadotropin-releasing hormone neuronal cells

Myosin X (Myo X), an unconventional myosin with a tail homology 4-band 4.1/ezrin/radixin/moesin (MyTH4-FERM) tail, is expressed ubiquitously in various mammalian tissues. In addition to the full-length Myo X (Myo X FL), a headless form is synthesized in the brain. So far, little is known about the function of this motor-less Myo X. In this study, the role of the headless Myo X was investigated in immortalized gonadotropin-releasing hormone (GnRH) neuronal cells, NLT. NLT cells overexpressing the headless Myo X formed fewer focal adhesions and spread more slowly than the wild-type NLT cells and GFP-expressing NLT cells. In chemomigration assays, the NLT cells overexpressing the headless Myo X migrated shorter distances and had fewer migratory cells compared with the control NLT cells.     

Evidence of extensive lymphocyte death in sheep Peyer's patches. I. A comparison of lymphocyte production and export

The metaphase arrest technique was used to determine the rate at which cells divide in the Peyer's patches (PP) and the thymus of 5 to 8 wk old lambs. The metaphase indices of these tissues were determined by analyzing cell suspensions of tissues taken before and 1, 2, 3, and 4 hr after metaphase arrest was initiated with i.v. vincristine. The metaphase indices increased in both tissues at a linear rate, which provided an estimate of the rate at which cells entered mitosis and of the lymphocyte birth rate. The ileal PP had the highest lymphocyte birth rate, 2.8% of the lymphocytes entered mitosis each hour; the rate was lower in jejunal PP (1.0%/hr) and thymus (0.5%/hr). With these values and estimates of the lymphocyte content in all PP (1.45 X 10(11)) and in the thymus (1.71 X 10(11)), it was calculated that the hourly lymphocyte production by PP in a lamb was 3.61 X 10(9) cells, which is four to five times greater than for the thymus (0.82 X 10(9)). Lymphocyte production in PP could then be compared with the number of lymphocytes that emigrated from the small intestine. Newly produced cells leave PP via the intestinal lymph, which could be collected from the entire small intestine after removal of the mesenteric lymph nodes. Cells entered the lymph at a rate of 0.8 X 10(9)/hr, but the output fell rapidly during chronic lymphatic drainage, a procedure known to deplete long-lived recirculating cells. It was concluded that most of the cells in intestinal lymph were recirculating cells, and newly formed lymphocytes produced in PP probably account for less than 25% of the total or 0.2 X 10(9)/hr. It seems unlikely that emigration could occur at a rate comparable with the rate of production in the PP. At most, only 5% of the PP cells seemed destined to leave their site of production, and it is proposed that most die within the PP follicles. The high mortality rate associated with the production of large numbers of B lymphocytes in lamb PP seems likely to have a significant impact on the nature of the contribution that these tissues make to the immune system.     

Mutagenesis of phi X174 am3 cs70 incorporated into the genome of mouse L-cells

The objective of our work with phi X174 has been to develop a shuttle vector that can be used comparatively in bacterial cells, different types of mammalian cells, and possibly in the various tissues of transgenic mice, with a constant mechanism for detection and analysis of mutations independent of any host-cell type. Toward that end, we have efficiently rescued phi X174 am3 cs70 that is host-silent and stably integrated into the genome of mouse L-cells. The particular mouse L-cell line contains tandem arrays, single copies, and fragments of phi X that, upon restriction enzyme excision, can result in 5 potentially active copies per diploid genome. The excised phi X DNA is recovered by column chromatography, ligated, and transfected into highly competent spheroplasts. The Rescue Efficiency, defined as the number of viable phages produced out of the total number of potentially recoverable copies, is approx. 10(-3). The Recovery Ratio, defined as the Rescue Efficiency for chromosomally-integrated phage DNA divided by the Rescue Efficiency for phi X am3 cs70, is close to one. Mouse L-cells containing the integrated phi X174 am3 cs70 were treated with 20 mM ethyl methanesulfonate. The reversion frequency of am3 among progeny phages rescued from treated cells was 1.4 X 10(-5) (193 revertants in 1.4 X 10(7) phages). This is significantly higher than the 5.8 X 10(-7) reversion frequency of am3 (7 revertants in 1.2 X 10(7) phages) among progeny phages rescued from untreated cells.     

Biochemical and immunological characterization of an H2A variant from the mouse testis

A histone H2A variant, protein 'A', has been isolated and purified from the mouse testis. Amino acid composition analysis and electrophoretic properties indicate it to be apparently similar to H2A X X variant present in the mouse L1210 cells. Specific antibodies against protein 'A' have been generated in rabbits and used to study tissue and species distribution in mammals. Protein transfer experiments indicate the presence of antigenically similar proteins in somatic tissues of the mouse. Immunologically similar proteins were also detected in other mammalian testes. The data further indicate that protein A is antigenically distinct from the other members of the H2A family, H2A X 1, H2A X 2 and H2A X Z.     

Increased skewing of X chromosome inactivation with age in both blood and buccal cells

The X chromosome inactivation pattern in peripheral blood cells becomes more skewed after age 55, and a genetic effect on this age-related skewing has been reported. We investigated the effect of age on X inactivation phenotype in blood, buccal cells and tissue from duodenal biopsies in 80 females aged 19-90 years. The X inactivation pattern correlated positively with age in blood (r = 0.238, P = 0.034) and buccal cells (r = 0.260, P = 0.02). The mean degree of skewing was higher in the elderly (>/=55 years) than in the young (<55 years) in blood (70.1 and 63.5%, respectively, P = 0.013) and in buccal cells (64.7 and 59.0%, respectively, P = 0.004). Correlation of X inactivation between the different tissues was high in all tissues with a tendency to increase with age for blood and buccal cells (P = 0.082). None of the duodenal biopsies had a skewed X inactivation, and the mean degree of skewing was similar in the two age groups. The tendency for the same X chromosome to be the preferentially active X in both blood and buccal cells with advancing age is in agreement with a genetic effect on age-related skewing and indicates that genes other than those involved in hematopoiesis should be investigated in the search for genes contributing to age related skewing.     

Frequent derepression of G6PD and HPRT on the marsupial inactive X chromosome associated with cell proliferation in vitro

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 depressed. 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.     

Silence of the fathers: early X inactivation

X chromosome inactivation is the mammalian answer to the dilemma of dosage compensation between males and females. The study of this fascinating form of chromosome-wide gene regulation has yielded surprising insights into early development and cellular memory. In the past few months, three papers reported unexpected findings about the paternal X chromosome (X(p)). All three studies agree that the X(p) is imprinted to become inactive earlier than ever suspected during embryonic development. Although apparently incomplete, this early form of inactivation insures dosage compensation throughout development. Silencing of the X(p) persists in cells of extraembryonic tissues, but it is erased and followed by random X inactivation in cells of the embryo proper. These findings challenge several aspects of the current view of X inactivation during early development and may have profound impact on studies of pluripotency and epigenetics.     

Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture

Chicken blastodermal cells were cultured for 48 hr as explanted intact embryos, as dispersed cells in a monolayer, or with a confluent layer of mouse fibroblasts. The cells were then dispersed and injected into stage X (E-G&K) recipient embryos that were exposed to 600 rads of irradiation from a ⁶⁰Co source. Regardless of the conditions in which the cells were cultured, chimeras with contributions to both somatic tissues and the germline were observed. When blastodermal cells were co-cultured with mouse embryonic fibroblasts, significantly more somatic chimeras were observed and the proportion of feather follicles derived from donor cells was increased relative to that observed following the injection of cells derived from explanted embryos or monolayer cultures. Culture of blastodermal cells in any of the systems, however, yielded fewer chimeras that exhibited reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Contributions to the germline were observed at an equal frequency regardless of the conditions of culture, but were significantly reduced in comparison to the frequency and rate of germ-line transmission following injection of cells obtained directly from stage X (E-G&K) embryos. These data demonstrate that some cells retain the ability to contribute to germline and somatic tissues after 48 hr in culture and that the ability to contribute to the somatic and germline lineages is not retained equally. © 1996 Wiley-Liss, Inc.