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PubMed2001

Epigenetic mark sequence of the H19 gene in human sperm.

We have investigated the epigenetic mark in the human H19 gene. The H19 promoter is methylation-free in human sperm, but it is methylated in the paternally derived allele of most adult tissues. Consequently, the H19 gene is exclusively transcribed from the maternal allele. It was demonstrated that the differentially methylated region (DMR) located 2 kb upstream from mouse H19 is essential for the imprinting of H19. A 39 bp sequence in DMR has a high degree of similarity between humans, mice and rats. The highly conserved 15 bp core region of the consensus sequence contains four methylatable sites, and thus has been proposed as a potential imprinting mark region. In this study, fine epigenetic sequencing analysis was performed on the sperm DNA in comparison with other adult organs. Interestingly, the conserved sequence of the potential mark region was methylated in almost all the sperm genomes analyzed. Furthermore, the single dinucleotide CpG, whose methylation affects the accessibility of the element to CTCF, was methylated in the conserved core in the human sperm. These results suggest that the human core sequences may act as an imprinting center in the reciprocal monoallelic expression of H19.

Adult↗
PubMed1998

Epigenetic reprogramming of the human H19 gene in mouse embryonic cells does not erase the primary parental imprint.

BACKGROUND: Genomic imprinting in mammals is thought to result from epigenetic modifications to chromosomes during gametogenesis, which leads to differential allelic expression during development. There is a requirement for an appropriate experimental system to enable the analysis of the mechanisms of genomic imprinting during embryogenesis. RESULTS: To develop a novel in vitro system for studying the molecular basis of genomic imprinting, we constructed mouse cell lines containing either a paternal or maternal human chromosome 11, by microcell-mediated chromosome transfer. Allele-specific expression and DNA methylation studies revealed that the imprinting status of the human H19 gene was maintained in mouse A9 mono-chromosomal hybrids. Each parental human chromosome was introduced independently into mouse near-diploid immortal fibroblasts (m5S) and two embryonal carcinoma (EC) cell lines (OTF9-63 and P19). The paternal allele of human H19 remained in a repressed state in m5S cells, but was de-repressed in both EC cells. The paternal H19 allele was demethylated extensively in OTF9-63 cells, whereas the only alteration in P19 hybrids was de novo methylation on both alleles in the 3' region. Following in vitro differentiation, the expressed paternal H19 allele was selectively repressed in differentiated derivatives of EC hybrids. CONCLUSION: These results indicated that human imprint marks could function effectively in mouse cells, and that the imprinting process was epigenetically reprogrammed in embryonal carcinoma cells, without erasure of the primary imprint that marked the parental origin. Therefore, these mono-chromosomal hybrids could provide a valuable in vitro system to study the mechanisms involved in the regulation of imprinted gene expression.

Animals↗
PubMed1984

Nature and origin of the RNA associated with simian virus 40 large tumor antigen.

Simian virus 40 (SV40) large tumor (T) antigen isolated from mammalian cells undergoing lytic or transforming infection is associated with small RNA fragments ("T-antigen RNA") that are protected from nuclease digestion. The rather high complexity of the ribonuclease T1 fingerprints of T-antigen RNA suggested that it is mainly derived from cellular heterogeneous nuclear RNAs. In the present study, 5'-32P-labeled T-antigen RNA was hybridized to monkey, mouse, and human Alu and SV40 DNA, and the nucleotide sequence of 37 T1 oligonucleotides was determined. The results suggest that the bulk of T-antigen RNA is derived from noncoding, double-stranded, ordered regions of cellular heterogeneous nuclear RNAs that exhibit sequence homologies with interspersed repetitive elements of the cellular genome. The possible biological implications of these results are discussed.

Animals↗
PubMed1986

Human c-myb protooncogene: nucleotide sequence of cDNA and organization of the genomic locus.

We have isolated cDNA clones of the human c-myb mRNA that contain approximately 3.4 kilobases of the approximately 3.8-kilobase mRNA sequence. Nucleotide sequence analysis shows that the c-myb mRNA contains an open reading frame of 1920 nucleotides, which could encode a 72-kDa protein. The cDNA nucleotide sequence and the predicted amino acid sequence of the c-myb protein are highly homologous to the corresponding chicken and mouse proteins. In particular, a region toward the NH2 terminus of the protein containing a 3-fold tandem repeat of 51 residues is evolutionarily conserved and is the only region of homology with the Drosophila c-myb protein. This region may represent a functionally important structure, most likely the DNA-binding domain. cDNA clones have been used to isolate genomic clones and to define a preliminary intron/exon organization of the c-myb gene. Identification of 5' and 3' coding and noncoding exons indicates that the human c-myb locus spans a 40-kilobase region.

Amino Acid Sequence↗
PubMed2003

Growth restricted in vitro culture conditions alter the imprinted gene expression patterns of mouse embryonic stem cells.

Embryonic stem (ES) cell-derived clones and chimeras are often associated with growth abnormalities during fetal development, leading to the production of over/under-weight offspring that show elevated neonatal mortality and morbidity. Due to the role played by imprinted genes in controlling fetal growth, much of the blame is pointed at improper epigenetic reprogramming of cells used in the procedures. We have analyzed the expression pattern of two growth regulatory imprinted genes, namely insulin like growth factor II (Igf2) and H19, in mouse ES cells cultured under growth restricted conditions and after in vitro aging. Culture of cells with serum-depleted media (starvation) and at high cell density (confluence) increased the expression of both imprinted genes and led to aberrant methylation profiles of differentially methylated regions in key regulatory sites of Igf2 and H19. These findings confirm that growth constrained cultures of ES cells are associated with alterations to methylation of the regulatory domains and the expression patterns of imprinted genes, suggesting a possible role of epigenetic factors in the loss of developmental potential.

Animals↗
PubMed2002

A human H19 transgene exhibits impaired paternal-specific imprint acquisition and maintenance in mice.

Genomic imprinting, the differential expression of autosomal genes based on their parent of origin, is observed in all eutherian mammals that have been examined. In most instances the genes that are imprinted in one species are imprinted in others as well, suggesting that imprinting predated eutherian radiation. For example, the RNA-coding H19 gene is repressed upon paternal inheritance in all species examined to date. Thus, it is surprising that there is remarkably little sequence conservation among the cis-acting DNA regulatory elements that are required for imprinting of H19 and the tightly linked Igf2 gene. The most conserved characteristic in the imprinting control region (ICR) is the presence of multiple binding sites for the zinc finger protein CTCF, raising the possibility that CTCF binding might be sufficient for the reciprocal imprinting of H19 and Igf2. To investigate whether a human H19 transgene, harboring seven CTCF sites, is correctly recognized and imprinted in the mouse, a 100 kb transgene containing the human H19 gene was introduced into the mouse germline. The human transgene was specifically methylated after passage through the male germline in a copy number-dependent manner, but the methylation was unstable, undergoing progressive loss during development. Consequently, the transgene was highly expressed upon both maternal and paternal inheritance. These results argue that the signals for both the acquisition and maintenance of methylation imprinting are diverging rapidly.

Animals↗
PubMed2000

Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo.

The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.

Animals↗
PubMed1997

Xist-deficient mice are defective in dosage compensation but not spermatogenesis.

The X-linked Xist gene encodes a large untranslated RNA that has been implicated in mammalian dosage compensation and in spermatogenesis. To investigate the function of the Xist gene product, we have generated male and female mice that carry a deletion in the structural gene but maintain a functional Xist promoter. Mutant males were healthy and fertile. Females that inherited the mutation from their mothers were also normal and had the wild-type paternal X chromosome inactive in every cell. In contrast to maternal transmission, females that carry the mutation on the paternal X chromosome were severely growth-retarded and died early in embryogenesis. The wild-type maternal X chromosome was inactive in every cell of the growth-retarded embryo proper, whereas both X chromosomes were expressed in the mutant female trophoblast where X inactivation is imprinted. However, an XO mouse with a paternally inherited Xist mutation was healthy and appeared normal. The imprinted lethal phenotype of the mutant females is therefore due to the inability of extraembryonic tissue with two active X chromosomes to sustain the embryo. Our results indicate that the Xist RNA is required for female dosage compensation but plays no role in spermatogenesis.

Animals↗
PubMed2000

Genetics. Reprogramming X inactivation.

Inactivation of one of the two X chromosomes occurs in all cells of female adult mice so that genes are expressed from only one X chromosome. In a Perspective, Clerc and Avner describe an elegant series of experiments in mouse embryos cloned from adult and embryonic female cell nuclei (Eggan et al.) that reveal how the inactivation state of the X chromosomes is reprogrammed.

Animals↗
PubMed1998

Molecular evidence for distinct genotypes of monkey B virus (herpesvirus simiae) which are related to the macaque host species.

Although monkey B virus (herpesvirus simiae; BV) is common in all macaque species, fatal human infections appear to be associated with exposure to rhesus macaques (Macaca mulatta), suggesting that BV isolates from rhesus monkeys may be more lethal to nonmacaques than are BV strains indigenous to other macaque species. To determine if significant differences that would support this supposition exist among BV isolates, we compared multiple BV strains isolated from rhesus, cynomolgus, pigtail, and Japanese macaques. Antigenic analyses indicated that while the isolates were very closely related to one another, there are some antigenic determinants that are specific to BV isolates from different macaque species. Restriction enzyme digest patterns of viral DNA revealed marked similarities between rhesus and Japanese macaque isolates, while pigtail and cynomolgus macaque isolates had distinctive cleavage patterns. To further compare genetic diversity among BV isolates, DNA sequences from two regions of the viral genome containing genes that are conserved (UL27 and US6) and variable (US4 and US5) among primate alphaherpesviruses, as well as from two noncoding intergenic regions, were determined. From these sequence data and a phylogenetic analysis of them it was evident that while all isolates were closely related strains of BV, there were three distinct genotypes. The three BV genotypes were directly related to the macaque species of origin and were composed of (i) isolates from rhesus and Japanese macaques, (ii) cynomolgus monkey isolates, and (iii) isolates from pigtail macaques. This study demonstrates the existence of different BV genotypes which are related to the macaque host species and thus provides a molecular basis for the possible existence of BV isolates which vary in their levels of pathogenicity for nonmacaque species.

Animals↗
PubMed2003

Reverse genetics for crimean-congo hemorrhagic fever virus.

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family BUNYAVIRIDAE: The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family BUNYAVIRIDAE: This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.

Animals↗
PubMed1984

Upstream region required for regulated expression of the glucose-repressible SUC2 gene of Saccharomyces cerevisiae.

The SUC2 gene produces two mRNAs with different 5' ends that encode two forms of invertase. The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose repression (carbon catabolite repression), and the 1.8-kilobase mRNA encoding intracellular invertase is produced constitutively at low levels. To identify 5' noncoding sequences essential for regulated expression of SUC2, we constructed in vitro a series of deletions and inserted them into the yeast genome at the chromosomal SUC2 locus. Analysis of the effects of each deletion on SUC2 gene expression identified an upstream region required for derepression of secreted invertase synthesis. The 3' boundary of this region is near -418. The 5' boundary does not appear to be sharply defined, but lies ca. 100 base pairs upstream. A deletion extending from -418 to -140 allowed high-level derepression, indicating that no essential sequences lie between the upstream region and the TATA box at -133 and that the upstream region can be moved 279 base pairs closer to the transcriptional start site. Interactions between the deletions and several unlinked mutations affecting the regulation of SUC2 gene expression were examined. Sequences between -1,900 and -86 are dispensable for expression of the 1.8-kilobase mRNA.

Base Sequence↗
PubMed1998

Fluorescence in situ hybridization analysis of the replication properties of the myotonic dystrophy protein kinase (DMPK) gene region.

Myotonic dystrophy (DM) is caused by an expansion of a CTG repeat sequence in the 3' noncoding region of a protein kinase gene (DMPK) at 19q13.3. We used in situ hybridization to analyse the replication timing of the genomic region containing DMPK in fibroblasts and myoblasts from controls and myotonic dystrophy patients. In this method the relative proportion of singlet to doublet hybridization signals is used to infer the relative time of replication of specific loci or regions. Our results show that in cells from normal individuals approximately 65% of signals appear as doublets, indicating early replication. In DM patients with a number of CTG repeats ranging from about 600-1800 we observed a significant increase of singlet-doublets compared to the background level. These results suggest the existence of replication alternations and/or structural differences between the normal and mutant alleles induced by the presence of the DM mutation.

Alleles↗
PubMed2006

Assessment of genomic imprinting of SLC38A4, NNAT, NAP1L5, and H19 in cattle.

BACKGROUND: At present, few imprinted genes have been reported in cattle compared to human and mouse. Comparative expression analysis and imprinting status are powerful tools for investigating the biological significance of genomic imprinting and studying the regulation mechanisms of imprinted genes. The objective of this study was to assess the imprinting status and pattern of expression of the SLC38A4, NNAT, NAP1L5, and H19 genes in bovine tissues. RESULTS: A polymorphism-based approach was used to assess the imprinting status of four bovine genes in a total of 75 tissue types obtained from 12 fetuses and their dams. In contrast to mouse Slc38a4, which is imprinted in a tissue-specific manner, we found that SLC38A4 is not imprinted in cattle, and we found it expressed in all adult tissues examined. Two single nucleotide polymorphisms (SNPs) were identified in NNAT and used to distinguish between monoallelic and biallelic expression in fetal and adult tissues. The two transcripts of NNAT showed paternal expression like their orthologues in human and mouse. However, in contrast to human and mouse, NNAT was expressed in a wide range of tissues, both fetal and adult. Expression analysis of NAP1L5 in five heterozygous fetuses showed that the gene was paternally expressed in all examined tissues, in contrast to mouse where imprinting is tissue-specific. H19 was found to be maternally expressed like its orthologues in human, sheep, and mouse. CONCLUSION: This is the first report on the imprinting status of SLC38A4, NAP1L5, and on the expression patterns of the two transcripts of NNAT in cattle. It is of interest that the imprinting of NAP1L5, NNAT, and H19 appears to be conserved between mouse and cow, although the tissue distribution of expression differs. In contrast, the imprinting of SLC38A4 appears to be species-specific.

Amino Acid Transport System A↗
PubMed2000

X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation.

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.

Animals↗
PubMed2001

Regulation of imprinted X-chromosome inactivation in mice by Tsix.

In mammals, X-chromosome inactivation is imprinted in the extra-embryonic lineages with paternal X chromosome being preferentially inactivated. In this study, we investigate the role of Tsix, the antisense transcript from the Xist locus, in regulation of Xist expression and X-inactivation. We show that Tsix is transcribed from two putative promoters and its transcripts are processed. Expression of Tsix is first detected in blastocysts and is imprinted with only the maternal allele transcribed. The imprinted expression of Tsix persists in the extra-embryonic tissues after implantation, but is erased in embryonic tissues. To investigate the function of Tsix in X-inactivation, we disrupted Tsix by insertion of an IRESbetageo cassette in the second exon, which blocked transcripts from both promoters. While disruption of the paternal Tsix allele has no adverse effects on embryonic development, inheritance of a disrupted maternal allele results in ectopic Xist expression and early embryonic lethality, owing to inactivation of both X chromosomes in females and single X chromosome in males. Further, early developmental defects of female embryos with maternal transmission of Tsix mutation can be rescued by paternal inheritance of the Xist deletion. These results provide genetic evidence that Tsix plays a crucial role in maintaining Xist silencing in cis and in regulation of imprinted X-inactivation in the extra-embryonic tissues.

Animals↗