PubMed Health⌕ Search

Biomedical subjects

R Feil

Publications and source records attributed to R Feil.

At least 37 records · Page 2Linked to original sources
PubMed2000

Leaf development in Ricinus communis during drought stress: dynamics of growth processes, of cellular structure and of sink-source transition.

Dicot leaf growth is characterized by partly transient tip-to-base gradients of growth processes, structure and function. These gradients develop dynamically and interact with dynamically developing stress conditions like drought. In Ricinus communis plants growing under well-watered and drought conditions growth rates peaked during the late night and minimal values occurred in the late afternoon. During this diurnal course the leaf base always showed much higher rates than the leaf tip. The amplitude of this diurnal course decreased when leaves approached maturity and during drought stress without any significant alteration of the diurnal pattern and it increased during the first days after rewatering. Unique relationships between leaf size and cytological structure were observed. This provided the framework for the analysis of changes in assimilation, transpiration and dark respiration, chlorophyll, protein, carbohydrate, and amino acid concentrations, and of activities of sink-source-related enzymes at the leaf tip and base during leaf development in well-watered and drought-stressed plants. Gas exchange was dominated by physiological rather than by anatomical properties (stomatal density). Tip-to-base gradients in carbohydrate concentrations per dry weight and sink-source-related enzymes were absent, whereas significant gradients were found in amino acid concentrations per dry weight. During drought stress, growing leaves developed source function at smaller leaf size, before specific physiological adaptations to drought occurred. The relevance of the developmental status of individual leaves for the drought-stress response and of the structural changes for the biochemical composition changes is discussed.

Amino Acids↗
PubMed2000

Igf2 imprinting in development and disease.

Igf2 is one of the first imprinted genes discovered and occupies a centre stage in the study of imprinting. This is because it has dramatic effects on the control of fetal growth, it is involved in growth disorders and in cancer, it interacts with products of other imprinted genes, and its imprinting status is under complex regulation in a cluster of tightly linked imprinted genes. Here we review briefly the key features of Igf2 imprinting in normal development and in disease, and hope to show what a fascinating subject of study this gene and its biology provides.

Animals↗
PubMed1999

Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.

Chromatin can be analysed by assaying its sensitivity to DNase I or other nucleases in purified nuclei. Usually, this is performed by Southern analysis of genomic DNA extracted from nuclease-treated nuclei, a methodology that requires many cells. Applying restriction fragment length polymorphisms (RFLPs), this methodology has been used for parental allele-specific chromatin studies on imprinted mammalian genes. However, such allelic studies are limited by the availability of suitable RFLPs. We therefore developed an alternative, PCR and single strand conformation polymorphism (SSCP)-based assay with which allelic sensitivity to nucleases can be determined in virtually all localised regions that have nucleotide polymorphisms. We also demonstrate that analysis of DNase I sensitivity can be performed on permeabilised cells. Combining the two approaches, in the imprinted mouse U2af1-rs1 gene we analysed parental allele-specific chromatin conformation in limited numbers of cultured cells. We also applied the PCR-SSCP approach to assay allelic DNA methylation at specific restriction enzyme sites. In summary, we developed an allele-specific assay that should be useful for biochemical and developmental investigation of chromatin, in particular for studies on genomic imprinting and X-chromosome inactivation.

Alleles↗
PubMed1999

Genomic imprinting in mammals: an interplay between chromatin and DNA methylation?

Most imprinted loci have key regulatory elements that are methylated on only one of the parental chromosomes. For several of these 'differentially methylated regions', recent studies establish that the unmethylated chromosome has a specialized chromatin organization that is characterized by nuclease hypersensitivity. The novel data raise the question of whether specific proteins and associated chromatin features regulate the allele-specificity of DNA methylation at these imprinting control elements.

Alleles↗
PubMed1999

Engineering the mouse genome by site-specific recombination.

Site-specific recombination systems are powerful tools for introducing predetermined modifications into eukaryotic genomes. Recent advances allow the manipulation of chromosomal DNA in a spatially and temporally controlled manner in mice, offering unprecedented possibilities for studying mammalian genome function and for generating animal models for human diseases.

Animals↗
PubMed1999

Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene.

The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5'-neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5' to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function.

Age Factors↗
PubMed1998

A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor.

We have developed a new ligand-dependent chimeric recombinase (Cre-GRdex) by fusing the site-specific Cre recombinase to the ligand binding domain (LBD) of a mutant human glucocorticoid receptor (GRdex). The synthetic glucocorticoid receptor (GR) ligands dexamethasone, triamcinolone acetonide and RU38486efficiently induce recombinase activity in F9 murine embryonal carcinoma cells expressing constitutively Cre-GRdex. In contrast, no recombinase activity was detected in the absence of ligand or in the presence of the natural GR ligands corticosterone, cortisol or aldosterone. Moreover, physiological concentrations of these natural GR ligands do not affect Cre-GRdexrecombinase activity induced by dexamethasone. Thus, as previously shown using Cre-oestrogen receptor (ER) fusion proteins, Cre-GRdexmight be useful for achieving loxP site-directed mutagenesis in cultured cells and spatio-temporally controlled somatic cell mutagenesis in transgenic mice.

Aldosterone↗
PubMed1998

Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep.

Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthenogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theories of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have investigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic development in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnormalities are most likely caused by imprinted genes. We demonstrate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These observations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants.

Alleles↗
PubMed1998

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes.

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

Alleles↗
PubMed1997

Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2.

The mouse insulin-like growth factor 2 (Igf2) locus is a complex genomic region that produces multiple transcripts from alternative promoters. Expression at this locus is regulated by parental imprinting. However, despite the existence of putative imprinting control elements in the Igf2 upstream region, imprinted transcriptional repression is abolished by null mutations at the linked H19 locus. To clarify the extent to which the Igf2 upstream region contains autonomous imprinting control elements we have performed functional and comparative analyses of the region in the mouse and human. Here we report the existence of multiple, overlapping imprinted (maternally repressed) sense and antisense transcripts that are associated with a tandem repeat in the mouse Igf2 upstream region. Regions flanking the repeat exhibit tissue-specific parental allelic methylation patterns, suggesting the existence of tissue-specific control elements in the upstream region. Studies in H19 null mice indicate that both parental allelic methylation and monoallelic expression of the upstream transcripts depends on an intact H19 gene acting in cis. The homologous region in human IGF2 is structurally conserved, with the significant exception that it does not contain a tandem repeat. Our results support the proposal that tandem repeats act to target methylation to imprinted genetic loci.

Alleles↗
PubMed1997

Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases, each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), respectively, but are insensitive to 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis.

Amino Acid Sequence↗
PubMed1997

Parental chromosome-specific chromatin conformation in the imprinted U2af1-rs1 gene in the mouse.

The imprinted U2af1-rs1 gene on mouse chromosome 11 is expressed exclusively from the paternal allele. We found that U2af1-rs1 resides in a chromosomal domain that displays marked differences in chromatin conformation and DNA methylation between the parental chromosomes. Chromatin conformation was assayed in brain and liver, in fetuses, and in embryonic stem cells by sensitivity to nucleases in nuclei. In all these tissues, the unmethylated paternal chromosome is sensitive to DNase-I and MspI and has two DNase-I hypersensitive sites in the 5'-untranslated region. In brain and in differentiated stem cells, which display high levels of U2af1-rs1 expression, a paternal DNase-I hypersensitive site is also readily apparent in the promoter region. On the maternal chromosome, in contrast, the entire U2af1-rs1 gene and its promoter are highly resistant to DNase-I and MspI in all tissues analyzed and are fully methylated. No differential MNase sensitivity was detected in this imprinted domain. The parental chromosome-specific DNA methylation and chromatin conformation were also present in parthenogenetic and androgenetic cells and in tissues from animals maternally or paternally disomic for chromosome 11. This demonstrates that these parental chromosome-specific epigenotypes are independently established and maintained and provides no evidence for interallelic trans-sensing and counting mechanisms in U2af1-rs1.

Alleles↗
PubMed1996

Nucleotide sequence of a 28-kb mouse genomic region comprising the imprinted Igf2 gene.

The mouse insulin-like growth factor II gene (Igf2) is physically linked to the insulin II gene (Ins2) and both are subject to tissue-specific genomic imprinting. The paternal-specific expression of Igf2 has been associated with hypermethylation of some CpG sites in the 5' flanking region and in the body of the gene. As a first step in analyzing the structural features of this imprinted locus, we here report the complete nucleotide sequence of Igf2, including all introns and the intergenic region adjacent to Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80% overall identity with the corresponding rat sequence and has a high GC content of 52%. In addition to the known CpG island within the second Igf2 promoter, another island was identified approximately 2 kb 5' to the first exon. Other features of this locus include a 35-fold tandem repeat of an 11-bp sequence that overlaps Igf2 pseudo-exon 2, and a B2 repeat element in the intergenic region between Ins2 and Igf2. The GC-richness and the presence of CpG islands associated with tandem repeats are common features of imprinted genes and thus may play a role in the imprinting mechanism.

Animals↗
PubMed1996

Ligand-activated site-specific recombination in mice.

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.

Animals↗
PubMed1995

Functional cGMP-dependent protein kinase is phosphorylated in its catalytic domain at threonine-516.

The phosphorylation of threonine residues in the catalytic core of several protein kinases is important for the functional integrity of these enzymes. The corresponding residues of cGMP-dependent protein kinase I alpha (cGMP kinase) are Thr-514 and/or Thr-516. The in vivo phosphorylation and functional role of these residues was studied. cGMP kinase was overexpressed and purified as a catalytically active and inactive enzyme in Sf9 insect cells and in Escherichia coli, respectively. The enzymological and physicochemical properties of the Sf9 cGMP kinase were indistinguishable from that of the purified bovine lung enzyme. The cysteines of cGMP kinase including Cys-518 were labeled with vinylpyridine. Amino acid sequencing and mass spectroscopy of the labeled peptides showed that Thr-516 was phosphorylated in the enzyme purified from Sf9 cells but not in that from E. coli. The functional importance of phosphothreonine-516 was investigated by substitution of Thr-516 by alanine (T516A) or by glutamate (T516E). Expression in insect cells of the T516A mutant resulted in a protein lacking detectable kinase activity, whereas the T516E mutant retained basal phosphotransferase activity. In E. coli, the exchange of Thr-516 by glutamate did not lead to the synthesis of a catalytically active enzyme. These results demonstrate that phosphothreonine-516 of cGMP kinase is crucial for the formation of an enzymatically active protein kinase.

Amino Acid Sequence↗
PubMed1995

Chromatin structure and imprinting: developmental control of DNase-I sensitivity in the mouse insulin-like growth factor 2 gene.

The insulin-like growth factor 2 (Igf2) gene on distal mouse chromosome 7 is expressed predominantly from the paternal allele. In previous studies we identified two regions of paternal allele-specific methylation; one at approximately 3 kb upstream of promoter 1, and a second in the 3', coding portion of the gene. The 3' region is methylated in an expressing tissue (fetal liver), whereas in a non-expressing tissue (fetal brain), it is not methylated. By contrast, in the 5' region, the paternal allele is highly methylated in all tissues. Here, we have studied another characteristic of chromatin, namely, sensitivity to DNase-I and have focused our developmental analysis on the two differentially methylated regions of Igf2. In the upstream region, four clustered DNase-I hypersensitive sites (HSS) were detected in embryonic stem (ES) cells and in midgestation embryos, but not in neonatal liver or brain. In promoter 1 (P1), at approximately 0.3 kb upstream of exon 1, we detected a tissue-specific HSS that was present in neonatal liver, in which P1 is active, but was absent in ES cells, the embryo, and in neonatal brain. No DNase-I HSS were detected in the 3' differentially methylated region of Igf2. In all these regions, we did not detect differences in DNase-I sensitivity between the parental chromosomes. These results establish major developmental and tissue-specific control of chromatin in the Igf2 locus. The presence of the HSS upstream of Igf2 precedes transcriptional activation of the Igf2 gene and may be indicative of a promoter for another transcript that is transcribed in the opposite direction. The HSS in P1 is largely liver-specific; this promoter therefore is differently regulated than the more general fetal promoters P2 and P3. Whereas methylation can be allele-specific, presumably reflecting the gene imprint, the nuclease sensitivity, as detected by our assay, is not. These results, taken together with previous observations, reveal developmental and tissue-specific complexity in the expression of the parental imprint at the level of chromatin and transcription. We propose that epigenetic features of tissue-specific control and of the control of allelic expression are intricately linked.

Alleles↗