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At least 1,603 records · Page 89Linked to original sources
PubMed1999

Acquisition of the H19 methylation imprint occurs differentially on the parental alleles during spermatogenesis.

The imprinted mouse H19 gene is hypomethylated on the expressed maternal allele and hypermethylated on the silent paternal allele. A 2-kb region of differential methylation located from -2 to -4 kb relative to the H19 transcriptional start site has been proposed to act as the imprinting mark since hypermethylation in this region is inherited from sperm and retained on the paternal allele throughout development. Here, we describe a temporal analysis of the methylation patterns at the H19 locus during postnatal male germ cell development. The 2-kb region is methylated on the paternal allele throughout spermatogenesis, suggesting that methylation is acquired in this region prior to the resumption of mitosis in postnatal male mice. Likewise, more than half of the maternal alleles are hypermethylated prior to the resumption of mitosis. However, the remaining maternal alleles are not hypermethylated until the completion of meiosis I, indicating that de novo methylation in this region is a continuous process. Sequences proximal to the H19 promoter, which are methylated in spermatozoa and on the paternal allele in somatic cells, are differentially methylated in diploid, mitotic spermatogonia. The maternal allele becomes hypermethylated in this region during meiotic prophase. Thus, the parental H19 alleles acquire methylation differentially in the male germline.

Alleles↗
PubMed2001

Imprinting status of 11p15 genes in Beckwith-Wiedemann syndrome patients with CDKN1C mutations.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by somatic overgrowth, congenital malformations, and predisposition to childhood tumors. Aberrant expression of multiple imprinted genes, including H19, IGF2, KCNQ1OT1, and CDKN1C, has been observed in BWS patients. It has been estimated that mutations in CDKN1C occur in 12-17% of BWS patients. We have screened 10 autosomal dominant pedigrees and 65 sporadic BWS cases by PCR/heteroduplex analysis and DNA sequencing and have identified four mutations, two of which were associated with biallelic IGF2 expression and normal H19 and KCNQ1OT1 imprinting. One patient demonstrated phenotypic expression of paternally transmitted mutation in this maternally expressed gene, a second proband is the child of one of a pair of monozygotic twin females who carry the mutation de novo, and a third patient exhibited unusual skeletal changes more commonly found in other overgrowth syndromes. When considered with other studies published to date, this work reveals the frequency of CDKN1C mutations in BWS to be only 4.9%. This is the first report of an analysis of the imprinting status of genes in the 11p15 region where CDKN1C mutations were associated with loss of IGF2 imprinting and maintenance of H19 and KCNQ1OT1 imprinting.

Amino Acid Sequence↗
PubMed1996

Three-exon structure of the gene encoding the rat prion protein and its expression in tissues.

The prion protein (PrP), encoded by a chromosomal gene, is associated with development of the neurodegeneration of prion-induced diseases. Since determination of the complete structure of the gene encoding PrP is important for understanding gene expression in the central nervous system (CNS), the nucleotide (nt) sequence of the isolated whole gene encoding rat PrP (raPrP) was determined. The rat PrP gene (raPrP) spans 16 kilobases (kb) of the rat genome and contains three exons of 19-47 base pairs (bp), 98 bp, and 2 kb separated by two introns of 2.2 kb and 11 kb. The first and second exons are noncoding, while the third exon contains a short 5' untranslated region, the entire 762-bp open reading frame (ORF), and a 3' untranslated region. The putative raPrP promoter in the 5' flanking region contains putative Sp1, AP-1, and AP-2 binding sites without a consensus TATA box. This TATA box-deficient feature, coupled with the presence of a high G+C content and Sp1-binding sites in the raPrP promoter, characterizes it as a housekeeping gene. Analysis of the raPrP cDNA 5'-end showed that raPrP mRNA transcription was initiated at multiple sites. Northern blot analysis showed that the levels of raPrP mRNA varied among rat tissues, with the highest levels found in the brain and placenta. This determination of raPrP nt sequences, including the introns and the 5' and 3' flanking regions, may make it possible to elucidate cis-acting elements that regulate the expression of this gene in different tissues and cell lines.

Amino Acid Sequence↗
PubMed2003

Biallelic expression of HRAS and MUCDHL in human and mouse.

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.

Alleles↗
PubMed1991

Molecular cloning of the thymus-specific parvalbumin known as avian thymic hormone: isolation of a full length cDNA and expression of the recombinant protein in Escherichia coli.

The complete coding sequence of the thymus-specific parvalbumin called avian thymic hormone (ATH) has been cloned into Escherichia coli. The translated amino acid sequence was found to be identical to the sequence of map turtle parvalbumin at 90 of 108 positions. Northern blot analysis of thymic RNA indicated a transcript length of approximately 1050 bp. However, the ATH cDNA probe failed to hybridize to poly(A)+ RNA from chicken leg muscle, a further indication that avian thymic hormone is distinct from the muscle-associated parvalbumin previously isolated from chicken. Southern analysis of chicken genomic DNA suggests the presence of a single copy of the ATH gene, and the absence of hybridization between an ATH cDNA fragment and genomic DNA from rat and rabbit is confirmatory evidence that ATH expression is restricted to avian species. One of the full length ATH cDNA clones harbored an insert that lacked all 5' noncoding sequences. This cDNA was inserted without further alteration into the prokaryotic expression vector, pKK223-3. The resulting construction, which contains eleven base pairs between the Shine-Dalgarno sequence and the initiation codon, affords reasonably high levels of expression in E. coli. In most respects, recombinant ATH mimics the tissue-derived protein, retaining a similarly high affinity for Ca2+ ion (KCa = 14 +/- 5 nM). However, in contrast to ATH isolated from chicken thymus tissue, the N-terminal alanine of recombinant ATH is unacetylated. As a result, the isoelectric point is shifted upward from 4.3 to approximately 4.8.

Amino Acid Sequence↗
PubMed1989

Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses.

The genome sequence of tick-borne encephalitis (TBE) virus (Western subtype vaccine strain Neudoerfl) was determined. This extends the previously published sequence of the structural proteins to the nonstructural protein region and noncoding sequences at the 5'- and 3'-termini. The amino-termini of the individual proteins were assigned by comparison with other flavivirus sequences. Amino acid homology calculations between TBE virus and mosquito-borne flaviviruses were performed for all nonstructural proteins. An evolutionary tree based on protein NS1 is presented that reveals the molecular basis of relationships among flaviviruses. Tick-borne and mosquito-borne flaviviruses share a common hydrophilicity profile and also other features of their primary sequences, such as the presumably functional Gly-Asp-Asp sequence element within protein NS5. Other characteristics, such as the potential N-glycosylation sites of protein NS1 and a potential proteolytic cleavage site within protein NS4B, are conserved within the mosquito-borne group, but differ in the TBE virus sequence.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence.

We have cloned and determined the nucleotide sequences of the seventh gene of the Miyahara strain of mumps virus (MuV) encoding the L protein. The L gene is 6925 nucleotides in length and contains a single long open reading frame which is capable of coding for a protein of 2261 amino acids with a calculated molecular weight of 256,571 Da. The deduced amino acid sequence of the L protein of MuV showed significant homology with those of six other paramyxoviruses, human parainfluenza type 2 virus, Newcastle disease virus, Sendai virus, measles virus, human parainfluenza type 3 virus, and human respiratory syncytial virus. The predicted MuV L protein contained distinct elements thought to be essential for RNA polymerase activity. A noncoding sequence of 24 nucleotides downstream of the presumed polyadenylation site of the L gene showed significant complementarity with the leader sequence composed of 55 nucleotide at the 3' end of the genomic RNA.

Amino Acid Sequence↗
PubMed1993

The leu-1 gene of Neurospora crassa: nucleotide and deduced amino acid sequence comparisons.

The Neurospora crassa leu-1 gene encodes beta-isopropylmalate dehydrogenase (IPMDH; EC 1.1.1.85), an enzyme in the leucine biosynthetic pathway. We determined the nucleotide sequence of the entire leu-1 gene and of four independent cDNA clones. By comparing the genomic and cDNA sequences, four introns were identified in the 5' portion of the gene and a single open reading frame was established. One of the introns is located within the 5'-noncoding region of the transcript. The deduced amino acid sequence encoded by leu-1 was aligned with that of the homologous yeast enzyme and extensive sequence identity was uncovered. The lesion present in a conventional leu-1 mutant was identified as the insertion of a single base pair.

3-Isopropylmalate Dehydrogenase↗
PubMed2025

Genetic and chromatin regulation of Pvt1 monoallelic expression.

While most genes are equivalently expressed on both alleles, genes with random monoallelic expression (RME) stably maintain expression from only one allele, but the mechanisms and consequences of RME remain unclear. We performed allele-specific RNA sequencing (RNA-seq) on ∼100 F1 hybrid neural progenitor cell (NPC) clonal lines to reveal the extent of autosomal RME (aRME). Of the 287 aRME genes, Pvt1, an oncogenic long non-coding RNA, is an aRME with a genetic bias. In the absence of genetic differences, Pvt1 undergoes balanced aRME. Pvt1 monoallelic expression is maintained by allele-specific active and repressive histone modifications, opposed to DNA methylation. Additionally, we provide a two-step mechanism for the initiation of aRME and demonstrate that Pvt1 monoallelic expression results in a growth phenotype due to the interplay with Myc. These findings provide insight into how genetic differences can skew a stochastic process, resulting in monoallelic expression with a phenotypic consequence in early development.

Chromatin↗
PubMed2004

Analysis of sex differences in EGC imprinting.

Sex-specific differences are apparent in the methylation patterns of H19 and Igf2 imprinted genes in embryonic germ cells (EGCs) derived from 11.5 or 12.5 days post coitum (dpc) primordial germ cells (PGCs). Here we studied whether these differences are associated either with the sex chromosome constitution of the EGCs or with the sex of the genital ridge (testis versus ovary) from which the PGCs were isolated. For this purpose we derived pluripotent EGC lines from sex-reversed embryos, either XY embryos deleted for Sry (XY(Tdym1)) or XX embryos carrying an Sry transgene. Southern blotting of the EGC DNA was used to analyze the differentially methylated regions of Igf2 and H19. The analysis revealed that both genes were more methylated in EGCs with an XY sex chromosome constitution than in those with an XX sex chromosome constitution, irrespective of the phenotypic sex of the genital ridge from which the EGCs had been derived. We conclude that the sex-specific methylation is intrinsic and cell-autonomous, and is not due to any influence of the genital ridge somatic cells upon the PGCs.

Animals↗
PubMed2004

Role of H19 3' sequences in controlling H19 and Igf2 imprinting and expression.

The regulation of H19 and Igf2 imprinting and expression depends on common elements. Using comparative analysis between human and mouse, we identified conserved regions 3' of the H19 transcription unit, including the H19/Igf2 endodermal enhancers and elements within a 4.2-kb domain between the H19 transcription unit and the enhancers. Transgene experiments implicate these elements in imprinting regulation. To establish whether they are required at the endogenous locus, first we replaced the endodermal enhancers with the alpha-fetoprotein endodermal enhancers (H19Afp). Second, we deleted the 4.2-kb region (H19delta4.2). Our analysis revealed that H19 and Igf2 imprinting and tissue-specific expression were maintained for both mutations, except for a slight reduction in paternal Igf2 expression from the H19Afp allele in liver. These results demonstrate that the H19 insulator can interact with heterologous enhancers to imprint Igf2. Furthermore, for H19, chromatin context or additional sequences possibly compensate for loss of conserved 3' elements.

3' Flanking Region↗
PubMed1998

Biallelic expression of all four IGF-II promoters and its association with increased methylation of H19 gene in human brain.

The human IGF-II gene is maternally imprinted in all tissues except adult liver and the choroid plexus/leptomeninges of the central nervous system where IGF-II is biallelically expressed. In human liver, it has recently been reported that this biallelic expression only involves the promoter P1 while the promoters P2-P4 direct IGF-II transcription monoallelically. To explore whether or not biallelic expression of the IGF-II promoters in human CNS displays the same pattern as in liver, we examined the allelic expression status of the four IGF-II promoters in human brain. We found that all four IGF-II promoters in human fetal and adult brain were expressed from both parental alleles. Furthermore, the levels of methylation of the 3' region of H19 gene in fetal brain were higher than those in other tissues with monoallelic expression of IGF-II. Since similar findings have been reported in Wilms' tumor, these data suggest a similar mechanism may be responsible for loss of imprinting of IGF-II in normal brain and Wilms' tumor.

Adult↗
PubMed1996

Molecular cloning and characterisation of the mouse preprogalanin gene.

Using a probe obtained by PCR amplification from mouse genomic DNA, a genomic clone was isolated covering the entire mouse preprogalanin gene. The mouse gene has an exon:intron organisation very similar to that of the rat and human genes. The first exon is noncoding while exons 2-5 carry the coding region. Exon 6 also encodes the stop codon and a polyadenylation signal. The deduced amino-acid sequence of mouse preprogalanin is 94% and 68% identical to the rat and human peptide, respectively. The amino-acid sequence of mouse galanin was confirmed by RT-PCR amplification of mouse brain RNA. The cloning of the mouse galanin gene should allow elucidation of the regulatory characteristics of its promoter and facilitate transgenic approaches to the analysis of galanin gene function in this species.

Amino Acid Sequence↗
PubMed1999

The genotype and epigenotype synergize to diversify the spatial pattern of expression of the imprinted H19 gene.

Little is known of how the genetic background effects the phenomenon of genomic imprinting. The H19 gene belongs to a cluster of imprinted genes on human chromosome 11. Here we show that the alternative splicing of a human H19 transcript is genotype-specific. Moreover, this variant transcript, which lacks exon 4, is either not found at all, is widely expressed or is confined to extra-villous cytotrophoblasts in first trimester placenta, depending on a combination of the genotype and the sex of the transmitting parent.

Alleles↗
PubMed2000

Maternal-specific footprints at putative CTCF sites in the H19 imprinting control region give evidence for insulator function.

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 (Igf2) gene and the closely linked H19 gene are regulated by an intervening 2 kb imprinting control region (ICR), which displays parentspecific differential DNA methylation [1] [2]. Four 21 bp repeats are embedded within the ICR and are conserved in the putative ICR of human and rat Igf2 and H19, suggesting that the repeats have a function [3] [4]. Here, we report that prominent DNA footprints were found in vivo on the unmethylated maternal ICR at all four 21 bp repeats, demonstrating the presence of protein binding. The methylated paternal ICR displayed no footprints. Significantly, the maternal-specific footprints were localized to putative binding sites for CTCF, a highly conserved zinc-finger DNA-binding protein with multiple roles in gene regulation including that of chromatin insulator function [5] [6]. These results strongly suggest that the maternal ICR functions as an insulator element in regulating mutually exclusive expression of Igf2 and H19 in cis.

Animals↗