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At least 685 records · Page 38Linked to original sources
PubMed2000

X inactivation-specific transcript expression in mouse oocytes and zygotes.

Expression of the X inactivation-specific transcript (XIST:) gene has previously been shown by reverse transcription-polymerase chain reaction (RT-PCR) to be present at the 4-cell stage of female mouse embryos. This early expression, which is followed by X inactivation in the extra-embryonic tissues, is maternally imprinted. By the blastocyst stage, as the embryonic lineages begin to form, the imprint is lost and expression becomes random. By applying in-situ RT-PCR, we showed that XIST: is expressed even earlier in development, in unfertilized mouse oocytes as well as in pronuclei stage zygotes. Our data demonstrate XIST: expression in oocytes and suggest that XIST: transcripts may occur in both XX and XY zygotes. A difference in the pattern of expression (rod-like or rounded punctate signal) is found among pronuclear-stage embryos. Early expression is in agreement with findings reported in human embryos.

Animals↗
PubMed2002

Sex-chromosome linked gene expression in in-vitro produced bovine embryos.

The expression of XIST, G6PD, HPRT, ZFX and ZFY were investigated in in-vitro produced bovine embryos. Transcripts of these genes were assayed by RT-PCR in pools of pre-compaction stage embryos and sexed pools of morulae and blastocysts. The expression of XIST, G6PD, HPRT and ZFX in female and male morulae and blastocysts were compared using a semi-quantitative RT-PCR. G6PD, HPRT and ZFX transcripts were noted in all pre-compaction stage embryos and in female and male blastocysts. ZFY transcripts were detected in unsexed pools of 8-16-cell stage embryos and in male blastocysts. XIST transcripts were detected in unsexed pools at the 8-16 cell stage, in male and female morulae, and in female blastocysts. The level of XIST RNA was significantly higher in female morulae than in males. Levels of G6PD and HPRT RNA were also higher in female morulae and blastocysts than in males, but only G6PD levels were significantly different between the sexes. The expression of ZFX was also significantly higher in female than in male blastocysts. These results show sexually dimorphic expression of sex chromosome linked genes prior to the blastocyst stage in in-vitro produced bovine embryos.

Animals↗
PubMed2005

Development of a monkey model for the study of primate genomic imprinting.

An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2, known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres (ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene. Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipulation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or by ICSI. Muscle biopsies followed by RT-PCR and sequence analysis were performed in four heterozygous animals produced by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differentially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells of the monkey.

Alleles↗
PubMed2005

IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch.

Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith-Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVF-derived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn's advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.

Animals↗
PubMed2006

Genetic imprinting during impaired spermatogenesis.

Disorders in genetic imprinting are discussed as potential genetic risk in assisted reproduction technology (ART), where most of the natural selection mechanisms are bypassed. As currently only limited information about genomic imprinting in disruptive spermatogenesis is available, we analysed the imprinting state of the paternally methylated gene H19 in various germ cell populations derived from seminiferous tubules exhibiting impaired spermatogenesis. Different germ cell types were isolated by laser microdissection from human testicular paraffin sections. Although the methylation state of the maternally imprinted gene SNRPN was investigated by methylation-specific PCR (M-PCR) to establish the isolation method, methylation of H19 was analysed by a single-strand conformation-based method. Contamination by somatic Sertoli cells was excluded because of Sertoli cell-specific vimentin immunohistochemistry before germ cell laser microdissection. We demonstrate correct genetic imprints for H19 even in spermatogonia selected from seminiferous tubules exhibiting spermatogenic arrest at the level of spermatogonia, providing no evidence for incorrect genomic imprinting in spermatozoa from infertile men used for ICSI.

Autoantigens↗
PubMed1994

Creation of a deletion series of mouse YACs covering a 500 kb region around Xist.

Two mouse YACs, PA-2 and PA-3, contain the Xist gene and are 460 kb and 3.3 Mb long respectively. While PA-2 is non-chimeric, PA-3 contains a substantial proportion of non-contiguous DNA. As a prerequisite to functional studies of the role of this region in X inactivation, we have created a deletion series of YACs that are spaced at approximately 50 kb intervals and were able to eliminate the unwanted chimeric sequences in YAC PA-3. For this purpose, we have constructed mouse B1 fragmentation vectors based on those described for human Alu fragmentation. Having created this series of YAC deletion derivatives, we were able to eliminate efficiently the 10-15% aberrant YACs that arise during the course of a fragmentation experiment by assessing their marker content. The overlap and the opposite orientation of the two YAC inserts permitted the creation of deletions on both sides of the 500 kb region around Xist. The use of this series of YACs in a biological assay will help us define the extent of the sequences necessary to bring about X chromosome inactivation.

Animals↗
PubMed1997

Identification and characterization of the human XIST gene promoter: implications for models of X chromosome inactivation.

The XIST gene in both humans and mice is expressed exclusively from the inactive X chromosome and is required for X chromosome inactivation to occur early in development. In order to understand transcriptional regulation of the XIST gene, we have identified and characterized the human XIST promoter and two repeated DNA elements that modulate promoter activity. As determined by reporter gene constructs, the XIST minimal promoter is constitutively active at high levels in human male and female cell lines and in transgenic mice. We demonstrate that this promoter activity is dependent in vitro upon binding of the common transcription factors SP1, YY1 and TBP. We further identify two cis -acting repeated DNA sequences that influence reporter gene activity. First, DNA fragments containing a set of highly conserved repeats located within the 5'-end of XIST stimulate reporter activity 3-fold in transiently transfected cell lines. Second, a 450 bp alternating purine-pyrimidine repeat located 25 kb upstream of the XIST promoter partially suppresses promoter activity by approximately 70% in transient transfection assays. These results indicate that the XIST promoter is constitutively active and that critical steps in the X inactivation process must involve silencing of XIST on the active X chromosome by factors that interact with and/or recognize sequences located outside the minimal promoter.

Animals↗
PubMed1998

Induction of XIST expression from the human active X chromosome in mouse/human somatic cell hybrids by DNA demethylation.

X chromosome inactivation occurs early in mammalian development to transcriptionally silence one of the pair of X chromosomes in females. The XIST RNA, a large untranslated RNA that is expressed solely from the inactive X chromosome, is implicated in the process of inactivation. As previous studies have shown that the XIST gene is methylated on the active X chromosome, we have treated a mouse/human somatic cell hybrid retaining an active human X chromosome with demethylating agents to determine whether expression of the human XIST gene could be induced. Stable expression of XIST was observed after several rounds of demethylation and stability of XIST expression correlated with the loss of methylation at the three sites analysed. We conclude that methylation is sufficient to inhibit expression of the XIST gene in somatic cell hybrids. No loss of expression was detected for eight other X-linked genes from the active X chromosome that was expressing XIST , suggesting that additional developmental or species-specific factors are required for the inactivation process.

Animals↗
PubMed1998

Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension.

The ratio of two differentially replicating alleles is not constant during S phase. Using this fact, we have developed a method for determining allele-specific replication timing for alleles differing by at least a single base pair. Unsynchronized cells in tissue culture are first sorted into fractions based on DNA content as a measure of position in S phase. DNA is purified from each fraction and used for PCR with primers that bracket the allelic difference, amplifying both alleles. The ratio of alleles in the amplified product is then determined by a single nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Riggs,A.D. (1993) Methods Enzymol., 225, 344-351]. We report here use of this SNuPE-based method to analyze replication timing of two X-linked genes, Pgk-1 and Xist, as well as the autosomal gene Gabra-6. We have found that the two alleles of the Gabra-6 gene replicate synchronously, as expected; similarly, the active allele of the Pgk-1 gene on the active X chromosome (Xa) replicates early relative to the silent allele on the inactive X chromosome (Xi). In contrast, the expressed allele of the Xist gene, which is on the Xi, replicates late relative to the silent allele on the Xa.

Alleles↗
PubMed1998

Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts.

We report here a new, sensitive and versatile genomic sequencing method, which can be used for in vivo footprinting and studies of DNA adducts. Starting with mammalian genomic DNA, single-stranded products are made by repeated primer extension; these products are subjected to homopolymeric ribonucleotide tailing at the 3' termini with terminal deoxynucleotidyl transferase and then ligated to a double-stranded linker having a complementary 3' overhang, and used for PCR. This terminal transferase-dependent PCR (TDPCR) method can generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR). A UV photofootprint in the mouse Xist gene promoter can be easily detected using TDPCR. No special enzymes or chemical reagents are needed to convert DNA adducts into strand breaks. Any lesion that blocks primer extension should be detectable.

Animals↗
PubMed1999

Collection of mRNA-like non-coding RNAs.

In last few years much data has accumulated which shows that in different cells various RNA transcripts are synthesized. They lack protein coding capacity and do not produce mature protein. It seems that they work mainly or exclusively on the RNA level. Their function and mechanism of action is poorly understood. In this paper we have collected all known RNA transcript and prepared a database for further structural and functional studies. This is the first collection of the nucleotide sequences of RNAs of this kind. The data can be accessed via WWW at: http://www.man.poznan.pl/5SData/ncRNA/inde x.html

Animals↗
PubMed2000

Evidence for evolutionarily conserved secondary structure in the H19 tumor suppressor RNA.

The molecular basis for function of the mammalian H19 as a tumor suppressor is poorly understood. Large, conserved open reading frames (ORFs) are absent from both the human and mouse cDNAs, suggesting that it may act as an RNA. Contradicting earlier reports, however, recent studies have shown that the H19 transcript exists in polysomal form and is likely translated. To distinguish between possible functional roles for the gene product, we have characterized the sequence requirements for H19-mediated in vitro suppression of tumor cell clonogenicity and analyzed the sequence of the gene cloned from a range of mammals. A cDNA version of the human gene, lacking the unusually short introns characteristic of imprinted genes, is as effective as a genomic copy in blocking anchorage-independent growth by G401 cells. The first 710 nucleotides of the gene can be deleted with no effect on in vitro activity. Further truncations from either the 5'- or 3'-end, however, cause a loss of suppression of clonogenicity. Using conserved sequences within the H19 gene as PCR primers, genomic DNA fragments were amplified from a range of mammalian species that span the functional domain defined by deletion analysis. Sequences from cat, lynx, elephant, gopher and orangutan complement the previous database of sequences from human, mouse, rat and rabbit. Hypothetical translation of the resulting sequences shows an absence of conserved ORFs of any size. Free energy and covariational analysis of the RNA sequences was used to identify potential helical pairings within the H19 transcript. A set of 16 helices are supported by covariation (i.e. conservation of base pairing potential in the absence of primary sequence conservation). The predicted RNA pairings consist largely of local hairpins but also include several long range interactions that bridge the 5'- and 3'-ends of the functional domain. Given the evolutionary conservation of structure at the RNA level and the absence of conservation at the protein level, we presume that the functional product of the H19 gene is a structured RNA.

Animals↗
PubMed2001

The non-coding RNAs as riboregulators.

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro-behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.

Animals↗
PubMed2001

Quantitation of telomerase components and hTERT mRNA splicing patterns in immortal human cells.

Telomerase is a reverse transcriptase that adds telomeric repeats to chromosomal ends. In most normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to limited proliferative life-span. Telomerase reactivation is associated with cellular immortalization and is a frequent event during tumorigenesis. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein subunit [human telomerase reverse transcriptase (hTERT)] and a template RNA (hTR). hTR is constitutively expressed, while hTERT is almost universally absent in telomerase-negative cells. Although repression of telomerase is transcriptional in telomerase-negative cells, post-transcriptional and assembly processes are likely to play important roles in regulating telomerase activity in those that are telomerase-positive. The telomerase transcript can also be alternatively spliced into a variety of non-functional forms. To establish the quantitative relationships between telomerase activity and its various components, we determined the numbers of molecules of hTR and hTERT mRNA, and the levels of alternatively spliced hTERT mRNA variants in normal, in vitro immortalized and cancer cell lines. We report here that there is surprisingly little variation in the proportion of alternatively spliced forms of hTERT in different cell lines. The only variation observed occurred when a change in splicing to non-functional forms appeared in response to conditions that repress telomerase activity in IDH4 cells. We also found that most telomerase-positive cell lines only contain a few molecules of potentially functional hTERT mRNA, and there is a correlation between telomerase activity and the levels of both hTR and hTERT +alpha+beta mRNA.

Alternative Splicing↗
PubMed2002

Methylation-dependent silencing at the H19 imprinting control region by MeCP2.

Methylation of CpG dinucleotides is correlated with transcriptional repression of genes, including imprinted genes. In the case of the imprinted H19 gene, a 2 kb imprinting control region (ICR) is subject to differential methylation, as it is methylated only on the silenced paternal allele. This region has previously been shown to act as a silencer element at the endogenous locus. The proteins that bind at the H19 differentially methylated domain (DMD) and mediate transcriptional silencing have yet to be identified, although a family of proteins containing a methyl-CpG-binding domain (MBD), of which MeCP2 is the best characterised, are obvious candidates. MeCP2 can bind to a single methylated CpG dinucleotide through its MBD and also contains a transcriptional repression domain (TRD). The TRD interacts with Sin3a and histone deacetylases (HDACs) in vivo, forming a repressive complex. Here we show that MeCP2 is recruited to the H19 DMD in vivo and can silence a reporter gene regulated by the H19 DMD in a methylation-dependent manner. This repression can be alleviated by deletion of the TRD from MeCP2 or by inhibition of HDAC activity. These data indicate that transcriptional silencing from the H19 ICR involves recruitment of MeCP2 and presumably an associated protein complex with deacetylase activity. This complex may also be recruited to the ICR in vivo, resulting in a compact, repressive chromatin structure capable of silencing the paternal H19 allele.

Animals↗
PubMed2004

Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability.

Active cytoplasmic RNA localization depends on the attachment of RNA-binding proteins that dictate the destination of the RNA molecule. In this study, we used an electrophoretic mobility-shift assay in combination with equilibrium and kinetic analyses to characterize the assembly of the human zipcode-binding protein IMP1 on targets in the 3'-UTR from Igf-II mRNA and in H19 RNA. In both cases, two molecules of IMP1 bound to RNA by a sequential, cooperative mechanism, characterized by an initial fast step, followed by a slow second step. The first step created an obligatory assembly intermediate of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a 'locked' stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities within RNP granules and with the localization apparatus.

3' Untranslated Regions↗