Mosaic methylation of Xist gene before chromosome inactivation in undifferentiated female mouse embryonic stem and embryonic germ cells

Epigenetic modification is implicated in the choice of the X chromosome to be inactivated in the mouse. In order to gain more insight into the nature of such modification, we carried out a series of experiments using undifferentiated mouse cell lines as a model system. Not only the paternally derived X (XP) chromosome, but the maternally derived one (XM) was inactivated in the outer layer of the balloon-like cystic embryoid body probably corresponding to the yolk sac endoderm of the post-implantation embryo in which XP is preferentially inactivated. Hence, it is likely that the imprint responsible for the nonrandom XP inactivation in early mouse development has been erased or masked in female ES cells. CpG sites in the 5' region of the Xist gene were partially methylated in female ES and EG and parthenogenetic ES cell lines as in the female somatic cell in which the silent Xist allele on the active X is fully methylated, whereas the expressed allele on the inactive X is completely unmethylated. In the case of undifferentiated ES cells, however, methylation was not differential between two Xist alleles. This observation was supported by the demonstration that single-cell clones derived from female ES cell lines were not characterized by either allele specific Xist methylation or nonrandom X inactivation upon cell differentiation. Apparently these findings are at variance with the view that Xist expression and X inactivation are controlled by preemptive methylation in undifferentiated ES cells and probably in epiblast.     

Structure and expression of the mouse L23mrp gene downstream of the imprinted H19 gene: biallelic expression and lack of interaction with the H19 enhancers

Previous studies have identified populations of dopamine neurons in the midbrain that colocalize cholecystokinin some of which project to the nucleus accumbens and caudate-putamen. The contribution of dopamine-colocalized peptide to the total releasable pool of cholecystokinin in these brain regions was investigated using microdialysis. Dopamine, dihydroxyphenylacetic acid and cholecystokinin immunoreactive levels in dialysates of the posterior medial nucleus accumbens and medial caudate-putamen were determined following 6-hydroxydopamine lesions of the ventral tegmental area and substantia nigra or transection of the medial forebrain bundle. An 89-99% depletion in basal extracellular dihydroxyphenylacetic acid and an 87-99% decrease in veratridine-evoked extracellular dopamine levels was observed in the nucleus accumbens and caudate-putamen, 4 weeks after 6-hydroxydopamine lesion. No statistically significant difference was observed between lesioned and control animals in the basal or veratridine-evoked extracellular level of cholecystokinin immunoreactivity in either region. Similarly, transection of the medial forebrain bundle failed to significantly deplete the releasable pool of cholecystokinin immunoreactivity in the nucleus accumbens or caudate nucleus despite 89-99% depletions of dopamine and its metabolite. These data suggest that midbrain dopamine or non-dopaminergic cells are not the primary source of releasable cholecystokinin in the posterior medial nucleus accumbens and medial caudate-putamen measured by microdialysis.     

Vasculogenesis from embryonic bodies of murine embryonic stem cells transfected by Tgf-beta1 gene

Alzheimer's disease (AD) is a progressive disorder associated with disruption of neuronal function and neuronal loss. N-acetylaspartate (NAA) is a marker of neuronal content and can be assessed using proton (1H) magnetic resonance spectroscopy (MRS). We utilized 1H-MRS (two-dimensional chemical-shift imaging) to assess amplitudes and areas of NAA, as well as choline moieties (Cho), creatine (Cr) and myo-inositol (mI), in 15 AD patients compared with 14 control subjects. Voxels were classified as predominantly cortical gray matter (CGM), subcortical gray matter (SGM), or white matter (WM). Compared with control subjects, AD patients exhibited decreased NAA/Cho and NAA/Cr amplitudes, whereas an increase was observed in Cho/Cr and in amplitude ratios involving mI. Area ratios were significant in the same direction for NAA/Cho, NAA/Cr, mI/Cr and mI/NAA. No significant effects of tissue type were observed; however, significant group x tissue type interactions were noted for Cho/Cr and mI/Cr amplitudes. Our study confirms that 1H-MRS can identify distinct physicochemical alterations in AD patients, reflecting membrane changes and diminished neuronal function. These alterations can be used as longitudinal markers for the disease.     

Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.     

Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RADIO gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1- and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT- cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT- products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1- cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1- cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1- cells are repaired by illegitimate recombination.     

Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells

Mouse has become an increasingly important organism for modeling human diseases and for determining gene function in a mammalian context. Unfortunately, transposon-tagged mutagenesis, one of the most valuable tools for functional genomics, still is not available in this organism. On the other hand, it has long been speculated that members of the Tc1/mariner-like elements may be less dependent on host factors and, hence, can be introduced into heterologous organisms. However, this prediction has not been realized in mice. We report here the chromosomal transposition of the Sleeping Beauty (SB) element in mouse embryonic stem cells, providing evidence that it can be used as an in vivo mutagen in mice.     

Evidence for the involvement of the Gli gene family in embryonic mouse lung development

Murine Gli, Gli2, and Gli3 are zinc finger genes related to Drosophila cubitus interuptus, a component of the hedgehog signal transduction pathway. In the embryonic lung, all three Gli genes are strongly expressed at the pseudoglandular stage, in distinct but overlapping domains of the mesoderm. Expression of Gli and Gli3, but not of Gli2, is subsequently downregulated at the canalicular stage, coincident with a decline in the expression of sonic hedgehog (Shh) and the hedgehog receptor gene, patched (Ptc). Overexpression of Shh in the lung results in increased levels of Ptc mRNA. Gli, but not Gli2, is also upregulated, suggesting a differential involvement of the Gli genes in the regulation of Ptc by SHH during lung development. Gli3 is not upregulated by Shh overexpression. However, its importance for lung development is shown by the finding that Gli3XtJ embryos, homozygous for a mutation involving a deletion of the Gli3 gene, have a stereotypic pattern of abnormalities in lung morphogenesis. The pulmonary defects in these embryos, consisting of localized shape changes and size reductions, correlate with normal Gli3 expression. Thus, our data indicate that one of the Gli genes, Gli3, is essential for normal lung development, and that another, Gli, can be placed downstream of Shh signaling in the lung.     

Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging

A mechanism of aging is proposed for mammals and other vertebrates. In this mechanism, most somatic cells have inherent deficiencies in methylation metabolism with respect to their capacity to methylate DNA. This leads to incomplete DNA methylation in each cell cycle which, accumulated over many cell cycles, contributes to genetic instability, senescence and cancer. These proposed metabolic deficiencies are present from the time somatic cells are young, yet it is only after many cell divisions that deleterious effects are realized. In nature, most animals have reproduced or have been killed by predators or other environmental hazards before they can be greatly affected by these deficiencies. These deficiencies evolved in animals eating a balance of nutrients from nature. Evidence from the literature is reviewed which establishes that methylation is lost from the DNA of many mammalian somatic cells as they age both in vivo and in vitro, and that DNA methylation levels are influenced by factors, such as diet, that affect methylation metabolism. Partially correcting the proposed deficiencies is considered as a possible molecular mechanism by which caloric restriction extends lifespan. Other possible dietary and transgenic means to correct the proposed deficiencies and extend lifespan are discussed.     

Generation of radiation-induced deletion complexes in the mouse genome using embryonic stem cells

As the genetic and physical mapping stage of the Human Genome Project nears completion, the focus is shifting toward the development of technologies for high-throughput analysis of gene function. Whereas DNA sequencing will enable the assignment of presumed function to a large number of genes in mice and humans, it is clear that the great majority of genes will have to be evaluated in vivo to accurately assess their role in a complex organism. While gene targeting in mouse embryonic stem (ES) cells is the current method of choice for the characterization of gene function in mice, it remains relatively labor intensive and lacks the throughput required for analysis of genome function on a large scale. Alternative methods of efficient mutagenesis will clearly be required for this task. Chromosomal deletions are powerful tools in the genetic analysis of complex genomes, enabling the systematic identification and localization of functional units along defined chromosomal regions. Not only are deletions useful for the identification of genetic functions, but they serve as mapping reagents for existing mutations or traits. While their use has been an essential tool in Drosophila genetics, classical mutagenesis in mice has been logistically impractical for generating deletions. We have previously described an efficient method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. In this article, we detail the methodological aspects of this technology and describe the applications of chromosomal deletions for characterizing gene function in ways that make optimal use of the information generated by the first stage of the Genome Project.     

The mouse histone H1 genes: gene organization and differential regulation

There are six mouse histone H1 genes present in the histone gene cluster on mouse chromosome 13. These genes encode five histone H1 variants expressed in somatic cells, H1a to H1e, and the testis-specific H1t histone. Two of the genes that have not been assigned previously to the five somatic H1 subtypes have been identified as encoding the H1b and H1d subtypes. Three of the H1 genes, H1a, H1c and H1t, are present on an 80 kb segment of DNA that contains nine core histone genes. Two others, H1d and H1e, are present in a second patch, while the H1b gene is at least 500 kb away in a patch containing 14 core histone genes. The histone H1 genes are differentially expressed. All five genes for the somatic histone H1 proteins are expressed in exponentially growing cells. However, the levels of H1a, H1b and H1d mRNAs are greatly reduced in cells that are terminally differentiated or arrested in G0, while the H1c and H1e mRNAs continue to be expressed. In addition to the major RNA that ends at the stem-loop, the H1c gene expresses a longer, polyadenylated mRNA in differentiated cells, although in varying amounts. None of the other histone H1 genes encodes detectable amounts of polyadenylated mRNAs.     

Chromatin modification of imprinted H19 gene in mammalian spermatozoa

To date, most functional analysis studies have focused on the effects of treatment contingencies on specific targeted aberrant and alternative responses. In the current investigation, the main and collateral effects of the assessment and treatment of attention-maintained self-injury were assessed. Specifically, we evaluated the effects of noncontingent and contingent social attention on four categories of behavior: self-injury, a novel mand, preexisting prosocial responses (e.g., babbling and reaching out), and other aberrant responses (i.e., aggression and destruction). Results suggested that self-injury, prosocial responses, and other aberrant behaviors were within the same functional response class. Possible impact of these results when selecting mands for functional communication training is discussed.