FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes

X-chromosome inactivation and the size of the CGG repeat number are assumed to play a role in the clinical, physical, and behavioral phenotype of female carriers of a mutated FMR1 allele. In view of the tight relationship between replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-chromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 heterozygous for a mutated FMR1 allele with a trinucleotide repeat number varying from 58 to a few hundred, and eight homozygous for a wild-type allele. We have applied two-color fluorescence in situ hybridization (FISH) with FMR1 and X-chromosome α-satellite probes to interphase cells of the various genotypes: the α-satellite probe was used to distinguish between early replicating (active) and late replicating (inactive) X-chromosomes, and the FMR1 probe revealed the replication pattern of this locus. All samples, except one with a large trinucleotide expansion, showed an early replicating FMR1 allele on the active X-chromosome and a late replicating allele on the inactive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, regardless of repeat size. We conclude therefore that: (1) the FMR1 locus is subjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat size, replicate later than wild-type alleles on both the active and inactive X-chromosomes; and (3) the delaying effect of the trinucleotide expansion, even with a low repeat size, is superimposed on the delay in replication associated with X-inactivation. Electronic Publication     

Differentiation of interphase domains of the homologous human X-chromosomes

In accordance with molecular biology data reported elsewhere, homologous interphase X-chromosome territories differ greatly in the abundance of inactive condensed chromatin. On the other hand, a three dimensional FISH (3D FISH) method has revealed that domains of both inactive and active X-chromosome have similar volumes and similar maximum section areas (Smax). To solve this contradiction, we examined differences between homologous human interphase X-chromosome territories using two dimensional FISH (2D FISH) preparations of clustered PHA-stimulated lymphocytes. For obtaining such preparations, we developed a new technique to avoid a stage of hypotonic treatment of living cells, since this treatment levels the chromatin compactness degree. According to our 2D FISH data, the mean ratios of Smax for larger and smaller homologous X-chromosomes, calculated for individual flattened nuclei, were 1.83 +/- 0.08 and 2.02 +/- 0.09, respectively, for clumped cells and groups of loosely associated and separated lymphocytes. In comparison, the same ratio calculated for individual 3D nuclei of PHA-stimulated lymphocytes was 1.38 +/- 0.05 (Falk et al., 2002). Our findings give evidence for enrichment of inactive X-chromosomes by low stretchable condensed chromatin. In addition, these findings show that an active X is enriched by a high stretchable form of chromatin, whose content may distinctly vary from cell to cell.     

Chromatin structure of active and inactive human X chromosomes

Nuclei from a variety of human cell lines and tissues were digested with gradually increasing levels of DNase I. The DNA was then purified, treated with restriction enzymes and subjected to Southern blot hybridization using a cloned cDNA probe to 3-phosphoglycerate kinase (PGK) a housekeeping enzyme. At relatively high levels of DNase I, a specific, slightly sensitive site in chromatin sequences encoding PGK was observed in all of the cell types examined. This slightly sensitive site resides on the active X-chromosome since cell lines with increased numbers of inactive X-chromosomes do not show an increase in the region of chromatin which is sensitive. Except for this restricted region of enhanced sensitivity on the active X-chromosome, the data suggest that, for PGK encoding sequences, chromatin configurations on the active and inactive X-chromosomes are similar.     

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cytological differentiation of active from inactive X domains in interphase: implications for X chromosome inactivation

A fluorescence in situ hybridization method using a biotinylated DNA probe specific for the centromeric region of the human X chromosome was used to differentiate the genetically active from the inactive X in interphase cells. With this technique, we were able to interpret both the relative position and the degree of condensation of the X chromosomes within the nucleus. We first established the specificity of fluorescence labelling of the hybridized probe by comparing its location and appearance (either dense or diffuse) when associated with a sex chromatin body (SCB) in early passage normal human female fibroblasts. In these cells, where the presence of inactive X chromatin was verified by identification of a 4',6-diamidino-2-phenyl indole (DAPI)-positive SCB in 85% of the cells examined, the X chromatin fluorescence was always associated with the SCB. The signal was dense in structure in 98% and peripheral in location in 80% of the nuclei. A second type of signal, diffuse in form, was observed in 85% of the nuclei and presumably represents the location of the active X chromosome. It was located peripherally or centrally with equal frequency and was not associated with any identifiable nuclear component. This diffuse signal was the major type associated with human male fibroblasts. In rodent x human hybrid cells containing a human inactive X, the fluorescent signal was associated with an SCB-like structure in only 13% of the nuclei; it was dense in 66% of the nuclei and equally peripheral or central in location. This indicates an alteration in the interphase structure of the human inactive X chromosome in hybrid cells which may explain its known instability with respect to genetic activity in such systems.     

Insect sex chromosomes

A presumptive mechanism of X inactivation has been investigated by using tritiated uridine-induced chromosome aberrations to distinguish active from inactive X chromosome arms in the insect Gryllotalpa fossor. Previous work on therian mammals has shown that constitutive and facultative heterochromatin are less susceptible to breakage by ³H-Urd than euchromatin (active). The present study indicates that, irrespective of the presence of two X chromosomes in females, only one of these is affected as in males and that the total number of aberrations induced by ³H-Urd in both male and female Gryllotalpa is the same. This suggests that in the female only one arm of one X chromosome is active and that a facultative heterochromatinization of the homologous arm of the other X is operative coupled with the presence of constitutive heterochromatin in the second arm of both X chromosomes.     

Cd bands and centromeric function in dicentric chromosomes

Summary The Cd technique was applied to two cases of dicentric attached X chromosomes (XpXp and XqXq) and to cells from an established cell line of tumor origin (MaNo9) in which dicentrics with two active centromeres were present and dicentrics with one active and one inactive centromere. It was confirmed that the Cd technique discriminates between active and latent centromeres, and it was demonstrated that true dicentrics and dicentrics with one latent centromere can co-exist in the same cells. This indicates that the mechanism of centromere inactivation is a phenomenon that is specific to each chromosome and not generalized at the level of the individual cell.     

Analysis of methylation and distribution of CpG sequences on human active and inactive X chromosomes by in situ nick translation

In situ nick translation of fixed mitotic chromosomes after HpaII or MspI digestion allows us to detect different DNA methylation levels along chromosomes. We used this technique to analyse the methylation levels of CCGG sites in the active and inactive X chromosomes of female human cells. In addition, we analysed the distribution of these sites with respect to the banding pattern. Our data show that the inactive X, as a whole, is more methylated than the active one and that CCGG sequences are preferentially located on R-positive bands.     

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

Mammalian plasma membrane ecto-nucleoside triphosphate diphosphohydrolase 1, CD39, is not active intracellularly. The N-glycosylation state of CD39 correlates with surface activity and localization

CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosphohydrolase family. The active site for native CD39 is located on the outer surface of the cellular plasma membrane; however, it is not yet known at what stage this enzyme becomes active along the secretory pathway to the plasma membrane. In this study, sucrose density fractionations performed on CD39-transfected COS-7 cell membranes suggest that CD39 activity resides primarily in the plasma membrane. Furthermore, we have created recombinant, soluble versions of CD39, one that is secreted and others that are retained in the endoplasmic reticulum, to demonstrate that CD39 is not active until it reaches the plasma membrane both in yeast and COS-7 cells. Moreover, the secreted active soluble CD39 in COS-7 cells is found to receive a higher degree of N-glycan addition than the inactive form retained intracellularly. When COS-7 cells were treated with tunicamycin to prevent N-glycosylation, soluble CD39 was not detected in the extracellular medium and remained inactive intracellularly. Surface biotinylation analysis also revealed that surface-expressed wild type CD39 receives a higher degree of N-glycosylation than intracellular forms and that inhibition of N-glycosylation prevents its plasma membrane localization. In addition, both intact and digitonin-permeablized COS-7 cells transfected with CD39 possess similar ecto-ATPase activities, further supporting the conclusion that only surface-expressed CD39 is enzymatically active. All of these data suggest that intracellular CD39 is inactive and that only a fully glycosylated CD39 has apyrase activity and is localized at the cell surface.     

Pattern of chromosomal replication in synchronized lymphocytes

Summary The BrdUrd-Hoechst 33258-Giemsa technique was employed to study patterns of chromosomal replication in human lymphocytes synchronized by Methotrexate (MTX). It is proposed that in the presence of MTX, a major portion of the cell population is blocked in an advanced stage of the S-phase and not in the G₁/S border of the cell cycle. At this point, the replication of the chromosomal segments corresponding to the R-bands is terminated, and the replication of the G-bands and the inactive X-chromosomes is being initiated. The use of this method in the study of higher resolution patterns of chromosomal replications is proposed.     

Rearrangement of tryptophan residues in caspase-3 active site upon activation

Caspase-3, one of the major apoptotic proteins, is a cysteine protease and exists as an inactive zymogen in healthy cells. In this study, the dynamic nature of the rearrangements of two tryptophan residues (Trp 206 and Trp 214) in the active sites of caspase-3 during the activation was analyzed by measuring the fluorescence lifetimes. Significant changes in the lifetime occurred upon activation by the specific cleavage. In addition, two mutant proteins that have only one tryptophan residue also showed the similar changes. These data indicate that the activation of caspase-3 resulted in the reorganization of both tryptophan residues.     

Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver.

Activity can be induced in potentially active rabbit skeletal muscle phosphorylase monomers covalently bound to Sepharose by noncovalent interaction with soluble subunits carrying inactive pyridoxal 5'-phosphate analogs or even salicyladlehyde. These analogs are themselves incapable of reconstituting active holophorphorylase from apophosphorylase. Phosphorylases with one intrinsically inactive and one potentially active subunit have about one half of the activity of the native phosphorylase dimer. The usefulness of this technique for subunit complementation was demonstrated by forming hybrid phosphorylases with inactive Sepharose-bound rabbit skeletal muscle subunits containing pyridoxal 5'-phosphate monomethylester and soluble activatable frog muscle and rabbit liver phosphorylase monomers. The inactive Sepharose-bound subunit induced in each case activity in the soluble subunit. But whereas the inactive rabbit muscle phosphorylase subunit even transmitted its characteristic temperature dependence of the rate of the reaction to the frog muscle subunit, it could not propagate its control properties to the liver enzyme. Differences of hybrid phosphorylases are related to immunological and amino acid divergencies among the component enzymes.