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PubMed2001

Elucidation of the minimal sequence required to imprint H19 transgenes.

The imprinted mouse H19 gene exhibits maternal allele-specific expression and paternal allele-specific hypermethylation. We previously demonstrated that a 14-kb H19 minitransgene possessing 5' differentially methylated sequence recapitulates the endogenous H19 imprinting pattern when present as high-copy arrays. To investigate the minimal sequences that are sufficient for H19 transgene imprinting, we have tested new transgenes in mice. While transgenes harboring limited or no 3' H19 sequence indicate that multiple elements within the 8-kb 3' fragment are required for appropriate imprinting, transgenes incorporating 1.7 kb of additional 5' sequence mimic the endogenous H19 pattern, including proper imprinting of low-copy arrays. One of these imprinted lines had a single 15.7-kb transgene integrant. This is the smallest H19 transgene identified thus far to display imprinting properties characteristic of the endogenous gene, suggesting that all cis-acting elements required for H19 imprinting in endodermal tissues reside within the 15.7-kb transgenic sequence.

Alleles↗
PubMed2001

Homology between a 173-kb region from mouse chromosome 10, telomeric to the Ifng locus, and human chromosome 12q15.

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).

Animals↗
PubMed1996

Recent amplification of rat ID sequences.

The rodent identifier (ID) family of repeats has amplified at an extremely high rate in the rat genome in recent evolutionary time, resulting in 130,000 copies per haploid genome. Statistical analyses support the grouping of 119 individual rat ID elements into four major and three minor subfamilies based on six diagnostic nucleotide positions. The consensus sequence of the oldest subfamily is identical to the ID region of the rat BC1 RNA gene, suggesting that the BC1 gene has dominated the early amplification of rat ID elements. The other six subfamilies share at least one diagnostic mutation in relation to the BC1 gene and show much less nucleotide sequence divergence, indicating that the recent large amplification of rat ID elements has been driven by another lineage of master gene(s) in the rat genome. This is consistent with the formation of a new lineage of master elements for ID amplification in rat. The formation of most rat ID elements appears to have occurred during the past three million years based on the results that four out of five randomly chosen ID elements are present only in the genome of one rat species and not in other closely related species.

Animals↗
PubMed1992

Genetic code and phylogenetic origin of oomycetous mitochondria.

We sequenced the 3'-terminal part of the COX3 gene encoding cytochrome c oxidase subunit 3 from mitochondria of Phytophthora parasitica (phylum Oomycota, kingdom Protoctista). Comparison of the sequence with known COX3 genes revealed that UGG is used as a tryptophan codon in contrast to UGA in the mitochondrial codes of most organisms other than green plants. A very high AT mutation pressure operates on the mitochondrial genome of Phytophthora, as revealed by codon usage and by A+T content of noncoding regions, which seems paradoxical because AT pressure causes tryptophan codon reassignment from UGG to UGA in mitochondria of most species. The genetic code and other data suggest that mitochondria of Oomycota share a direct common ancestor with mitochondria of plants and that mitochondria of the ancestor of Planta and Oomycota were acquired in a second endosymbiotic event, which occurred later than the acquisition of mitochondria by other eukaryotes.

Amino Acid Sequence↗
PubMed2003

Controlling X-inactivation in mammals: what does the centre hold?

Controlling gene expression is one of the most fundamental task of living organisms, from prokaryotes to higher eukaryotes, in order to develop, grow, and reproduce in an ever changing environment. In many cases, the expression status of a given gene is controlled independently of that of its neighbours through localised cis DNA elements responsible for the recruitment of specific factors and enzymatic activities. However, in a growing number of cases, genomic regions including several genes have been shown to be regulated in a coordinated manner. X-chromosome inactivation, the dosage compensation mechanism encountered in mammals, is one of the most Striking example of such coordinated gene regulation. This process, which occurs at the chromosome-wide level, affecting many hundreds of genes, is under the control of a unique, cis acting region, termed the X-inactivation centre, whose complexity is just beginning to be unravelled.

Animals↗
PubMed2001

CpG methylation regulates the Igf2/H19 insulator.

The differentially methylated 5'-flank of the mouse H19 gene unidirectionally regulates the communication between enhancer elements and gene promoters and presumably represses maternal Igf2 expression in vivo [1-6]. The specific activation of the paternally inherited Igf2 allele has been proposed to involve methylation-mediated inactivation of the H19 insulator function during male germline development [1-4, 6]. Here, we addressed the role of methylation by inserting a methylated fragment of the H19-imprinting control region (ICR) into a nonmethylated episomal H19 minigene construct, followed by the transfection of ligation mixture into Hep3B cells. Individual clones were expanded and analyzed for genotype, methylation status, chromatin conformation, and insulator function. The results show that the methylated status of the H19 ICR could be propagated for several passages without spreading into the episomal vector. Moreover, the nuclease hypersensitive sites, which are typical for the maternally inherited H19 ICR allele [1], were absent on the methylated ICR, underscoring the suggestion that the methylation mark dictates parent of origin-specific chromatin conformations [1] that involve CTCF [2]. Finally, the insulator function was strongly attenuated in stably maintained episomes. Collectively, these results provide the first experimental support that the H19 insulator function is regulated by CpG methylation.

Alleles↗
PubMed1997

An imprinting element from the mouse H19 locus functions as a silencer in Drosophila.

Genomic imprinting as originally described in Sciara is displayed by many organisms. In mammals, X-inactivation and the parent-of-origin-specific silencing of imprinted genes are examples of this phenomenon. A heritable chromatin structural modification may be the critical mechanism in such instances of chromosome condensation and preferential gene inactivation. H19 is an imprinted gene in which the repressed paternal allele is hypermethylated and the compacted chromatin is relatively resistant to digestion by nucleases. In order to uncover underlying conserved epigenetic mechanisms we have introduced a mouse H19 transgene into Drosophila. We show here that a 1.2-kb H19 upstream sequence functions in cis as a parent-of-origin independent silencing element in Drosophila. Strikingly, this cis-acting element is located within an upstream region that is necessary for H19 imprinting in mice. These results suggest involvement of an evolutionary conserved mechanism in both genes silencing in Drosophila and imprinting in mice.

Animals↗
PubMed2005

Conserved noncoding sequences are selectively constrained and not mutation cold spots.

Noncoding genetic variants are likely to influence human biology and disease, but recognizing functional noncoding variants is difficult. Approximately 3% of noncoding sequence is conserved among distantly related mammals, suggesting that these evolutionarily conserved noncoding regions (CNCs) are selectively constrained and contain functional variation. However, CNCs could also merely represent regions with lower local mutation rates. Here we address this issue and show that CNCs are selectively constrained in humans by analyzing HapMap genotype data. Specifically, new (derived) alleles of SNPs within CNCs are rarer than new alleles in nonconserved regions (P = 3 x 10(-18)), indicating that evolutionary pressure has suppressed CNC-derived allele frequencies. Intronic CNCs and CNCs near genes show greater allele frequency shifts, with magnitudes comparable to those for missense variants. Thus, conserved noncoding variants are more likely to be functional. Allele frequency distributions highlight selectively constrained genomic regions that should be intensively surveyed for functionally important variation.

Conserved Sequence↗
PubMed2003

Identical rearranged forms of JC polyomavirus transcriptional control region in plasma and cerebrospinal fluid of acquired immunodeficiency syndrome patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by the human polyomavirus JC (JCV). JCV has a hypervariable noncoding transcriptional control region (TCR) that spans the origin of replication of the JCV genome through to the first ATG start codon for late gene transcription. The archetype form of TCR is frequently found in the urine and kidneys of healthy and immunocompromised subjects. However the rearranged forms, whose prototype is Mad-1, possibly generated by deletion and duplication of segments of the archetype sequence, are found in the brain and cerebrospinal fluid (CSF) of PML patients. In this study the authors compared JCV TCR detected in paired CSF, plasma, and urine samples of 11 acquired immunodeficiency syndrome (AIDS) patients affected by PML to try to determine where the rearranged JCV TCRs are selected. In one patient, it was also possible to amplify and sequence the TCR in the brain and lymphocytes. Moreover, in 5/11 patients, the CSF, plasma, and urine samples corresponding to 2 months after PML development were available; and in another patient, it was possible to sequence the TCR in plasma and lymphocytes sampled 8 months before the onset of PML. The presence of the same TCR sequences in all the CSF and plasma samples taken from individual patients could strengthen the hypothesis that the blood is a compartment where JCV may replicate and undergo rearrangement of the TCR. This further supports the hypothesis that JCV reaches the brain by a hematogenous route and indicates that the JCV TCR sequences detected in plasma could be used as an early marker of JCV pathogenicity before the clinical appearance of PML in immunocompromised patients.

AIDS-Related Opportunistic Infections↗
PubMed1998

The mouse H19 locus mediates a transition between imprinted and non-imprinted DNA replication patterns.

Genes subject to genomic imprinting generally occur in clusters of hundreds of kilobases. These domains exhibit several gamete of origin-dependent manifestations, including a pattern of asynchronous replication when studied by fluorescence in situ hybridization (FISH). We find a transition from asynchronous replication at the imprinted mouse H19 gene to synchronous replication at the downstream Rpl23 gene, the human homologue of which appears to be non-imprinted. Two-colour FISH demonstrates that this transition is due solely to a difference in replication timing between the upstream and downstream chromatin on the later-replicating (maternal) chromosome. This difference is lost in mice deleted for the H19 gene body and 9.9 kb of upstream DNA when this deletion is maternally inherited, with synchronous replication patterns extending over 110 kb upstream from the deleted area. No effect is seen when the deletion is paternally inherited. The presence of a boundary element in this region has been suggested by observations of position-independent expression of H19 -containing transgenes and the blocking of accessibility of downstream enhancers to the upstream Igf2 and Ins2 genes on the maternal chromosome. The FISH studies presented here demonstrate the insulation of replication patterns within the imprinted domain from downstream, non-imprinted chromatin, mediated by an element at the H19 locus which is subject to genomic imprinting.

Animals↗
PubMed1983

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end. This coding region is flanked by a 5'-terminal sequence of 54 nucleotides and a 3'-noncoding region of 166 nucleotides which includes the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common.

Amino Acid Sequence↗
PubMed1988

Sigma region located between C mu and C delta genes of human immunoglobulin heavy chain: possible involvement of tRNA-like structure in RNA splicing.

Noncoding regions within the cluster of immunoglobulin heavy chain constant genes in the human genome contained a number of repeats. In the mu-delta intron, two repeating units were contained. One 442-base-long fragment located JH-mu intron (defined as "sigma mu(sigma mu)") occupied the position in the mu-delta intron. The other 1166-base-long fragment located somewhere in front of S (class switch) region of C gamma gene was also found in the mu-delta intron. We defined the repeats in the mu-delta intron as "SIGMA (sigma)". The polarities of the longer repeats in the genome were opposite between the mu-delta intron and the upstreams of C gamma genes. These inverted copies (defined as sigma gamma 3 and sigma gamma 4), located 6 kb upstream of their respective C gamma's, were apparently transcribed in vitro, via RNA polymerase III and transcripts should have contained tRNA-like structures. Small DNA fragments capable of encoding tRNA-like structures were also found in corresponding regions of mouse Ig C gamma cluster.

Animals↗
PubMed2004

Genomic imprinting of H19 in naturally reproduced and cloned cattle.

Animals produced from assisted reproductive technologies suffer from developmental abnormalities and early fetal death at a higher frequency than that observed in those produced by natural breeding. These symptoms are reminiscent of imprinting disruptions in the human and mouse, suggesting the possibility of perturbations in the expression of imprinted genes such as biallelic expression or silencing. H19 is one of the imprinted genes first identified in mice and humans, but its sequence and imprinting status have not been determined in cattle. In the present study, we obtained the majority of the bovine H19 gene sequence (approximately 2311 base pairs), identified a single nucleotide polymorphism (SNP) in exon 5 and determined the frequencies of different alleles containing the SNP. Our analysis demonstrated that, in cattle produced by natural breeding, H19 was indeed imprinted as shown by either predominant or exclusive expression of the maternal allele. We also analyzed the imprinting pattern of H19 in organs of four animals produced by somatic cell nuclear transfer that died shortly after birth or had developed abnormalities that necessitated immediate killing at birth. Three out of four cloned animals showed biallelic expression of H19, supporting our hypothesis that imprinting disruption is present in cloned animals that suffered from developmental abnormalities at birth. Examination of the expression of H19 in the offspring of a cloned animal produced by artificial insemination showed that the imprinting pattern in this animal was indistinguishable from those of control animals, suggesting that either imprinting disruptions in cloned animals are corrected through natural reproduction or that they are not present in healthy cloned animals capable of undergoing natural reproduction.

Alleles↗
PubMed1999

Effect of loss of DNA mismatch repair on development of topotecan-, gemcitabine-, and paclitaxel-resistant variants after exposure to cisplatin.

Loss of DNA mismatch repair (MMR) causes genomic instability by markedly increasing the frequency of sporadic mutations in both coding and noncoding sequences. Little is known about how loss of MMR affects sensitivity to the mutagenic effect of chemotherapeutic agents. We wanted to determine how loss of MMR affects the ability of cisplatin, a known mutagen, to generate human tumor cell variants resistant to other drugs with which cisplatin is commonly combined in treatment regimens. We compared the ability of cisplatin to produce variants resistant to topotecan, gemcitabine, and paclitaxel in two pairs of MMR-proficient and -deficient cells that included sublines of the human colon carcinoma cell line HCT-116 and sublines of the human endometrial adenocarcinoma cell line HEC59. Cells were exposed to increasing concentrations of cisplatin for 1 h, and the surviving population was tested for the frequency of variants resistant to these single molecular target drugs 10 days later. The frequency of variants increased linearly with cisplatin concentration for all three drugs. Cisplatin was 2.6 +/- 0.3- (S.D.), 3.6 +/- 0.9-, and 2.3 +/- 0.1-fold more potent at producing topotecan-, gemcitabine-, and paclitaxel-resistant variants in the MMR-deficient than in the MMR-proficient HCT116 cells (P <.05 for all). Cisplatin was 1.4 +/- 0.3- and 1.4 +/- 0.4-fold more potent at generating topotecan- and gemcitabine-resistant variants in MMR-deficient HEC59 cells than in MMR-proficient HEC59+ch2 cells. Cisplatin was not more potent in generating paclitaxel-resistant variants in the MMR-deficient HEC59 cells. Spontaneous rates of generation of cells resistant to these three drugs were also measured in the HCT116 sublines. MMR-deficient HCT116 cells exhibited rates of generation of resistant variants that were 1.94- and 1.51-fold higher (P <.05) than those in the MMR-proficient cells for topotecan and gemcitabine, respectively; loss of MMR had no effect on the rate of generation of variants resistant to paclitaxel. We conclude that the loss of MMR increases the ability of cisplatin to generate variants resistant to topotecan, gemcitabine, and possibly paclitaxel and that MMR also plays a role in controlling the spontaneous rate of generation of variants resistant to topotecan and gemcitabine.

Antineoplastic Agents↗
PubMed1993

Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism.

The human hepatitis C virus (HCV) contains a long 5' noncoding region (5' NCR). Computer-assisted and biochemical analyses suggest that there is a complex secondary structure in this region that is comparable to the secondary structures that are found in picornaviruses (E.A. Brown, H. Zhang, L.-H. Ping, and S.M. Lemon, Nucleic Acids Res. 20:5041-5045, 1992). Previous in vitro studies suggest that the HCV 5' NCR plays an important role during translation (K. Tsukiyama-Kohara, N. Iizuka, M. Kohara, and A. Nomoto, J. Virol. 66:1476-1483, 1992). Dicistronic and monocistronic expression vectors, in vitro translation, RNA transfections, and deletion mutagenesis studies were utilized to demonstrate unambiguously that the HCV 5' NCR is involved in translational control. Our data strongly support the conclusion that an internal ribosome entry site exists within the 5' noncoding sequences proximal to the initiator AUG. Furthermore, our results suggest that the HCV genome is translated in a cap-independent manner and that the sequences immediately upstream of the initiator AUG are essential for internal ribosome entry site function during translation.

Base Sequence↗
PubMed2026

Differentiation-independent activation of HPV genome replication by the lncRNA DINO.

Human papillomaviruses (HPVs) rely on multiple host cell factors to replicate the viral genome, yet the contribution of host long non-coding RNAs (lncRNAs) to viral genome maintenance and amplification in the productive life cycle remains poorly understood. In this study, we show that the lncRNA damage-induced long non-coding RNA (DINO) is a driver of HPV DNA replication. DINO levels increase during keratinocyte differentiation, and ectopic expression of DINO promotes both HPV genome replication and the formation of replication foci, and this is independent of keratinocyte differentiation signals. Ectopic DINO expression increases select early viral transcript levels, including E1^E4, E1, and E2. Notably, DINO's subcellular localization is also context-dependent: during DNA damage, DINO is predominantly cytoplasmic, but during keratinocyte differentiation, nuclear retention is observed. This differential localization suggests that DINO has distinct functional roles in keratinocyte differentiation and HPV biology. Our findings highlight DINO as a lncRNA that promotes HPV genome replication and suggest that lncRNAs may play underappreciated roles in host-virus interactions. This work provides a foundation for further exploration of lncRNAs as potential therapeutic targets in HPV-associated diseases.IMPORTANCEHuman papillomaviruses (HPVs) are the causative agents of many anogenital tract and oral cancers, yet the host factors that trigger and support viral genome replication during the productive life cycle are incompletely understood. This study identifies the long non-coding RNA DINO as a host regulator that promotes HPV DNA replication, replication focus formation, and early viral gene expression independently of keratinocyte differentiation. We further show that DINO exhibits context-dependent subcellular localization, suggesting distinct functional roles in cellular stress responses and HPV biology. These findings reveal an underappreciated role for host lncRNAs in virus-host interactions and provide new insight into cellular pathways that support HPV genome replication.

Virus Replication↗