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At least 1,585 records · Page 88Linked to original sources
PubMed2006

Evidence for a microRNA expansion in the bilaterian ancestor.

Understanding how animal complexity has arisen and identifying the key genetic components of this process is a central goal of evolutionary developmental biology. The discovery of microRNAs (miRNAs) as key regulators of development has identified a new set of candidates for this role. microRNAs are small noncoding RNAs that regulate tissue-specific or temporal gene expression through base pairing with target mRNAs. The full extent of the evolutionary distribution of miRNAs is being revealed as more genomes are scrutinized. To explore the evolutionary origins of metazoan miRNAs, we searched the genomes of diverse animals occupying key phylogenetic positions for homologs of experimentally verified human, fly, and worm miRNAs. We identify 30 miRNAs conserved across bilaterians, almost double the previous estimate. We hypothesize that this larger than previously realized core set of miRNAs was already present in the ancestor of all Bilateria and likely had key roles in allowing the evolution of diverse specialist cell types, tissues, and complex morphology. In agreement with this hypothesis, we found only three, conserved miRNA families in the genome of the sea anemone Nematostella vectensis and no convincing family members in the genome of the demosponge Reniera sp. The dramatic expansion of the miRNA repertoire in bilaterians relative to sponges and cnidarians suggests that increased miRNA-mediated gene regulation accompanied the emergence of triploblastic organ-containing body plans.

Algorithms↗
PubMed2002

Transcription repair coupling factor: a very pushy enzyme.

An irreversibly stalled RNA polymerase (RNAP) in the coding region of an active gene blocks transcription and can lead to the loss of genome integrity. A recent study of the bacterial Mfd protein (Park et al., 2002 [Cell volume 109]) provides new insight into mechanisms that release RNAP from sites of arrest such as noncoding DNA lesions.

Animals↗
PubMed2001

Expression of VP5 of infectious pancreatic necrosis virus strain VR299 is initiated at the second in-frame start codon.

Infectious pancreatic necrosis virus (IPNV), a member of the BIRNAVIRIDAE: with two double-stranded RNA genome segments, encodes five proteins designated VP1 to VP5. To study the function of the 17 kDa nonstructural protein VP5 during virus replication several mutated IPNV genome segments A were constructed and included in a reverse genetics system for IPNV to obtain recombinant virus. Mutations between nt 68 and 85 or nt 94 and 103 in the noncoding region failed to yield viable virus. Only mutations located between nt 86 and 92 and downstream of nt 104 were tolerated, and viable virus could be generated. All IPNV generated showed no difference in replication compared with the wild-type IPNV, indicating that the absence of expression of VP5 did not influence virus growth in vitro. Furthermore, the results presented here indicate that initiation of translation of VP5 occurs at position 113, the second in-frame start codon.

Amino Acid Sequence↗
PubMed2002

MicroRNAs in plants.

MicroRNAs (miRNAs) are an extensive class of ~22-nucleotide noncoding RNAs thought to regulate gene expression in metazoans. We find that miRNAs are also present in plants, indicating that this class of noncoding RNA arose early in eukaryotic evolution. In this paper 16 Arabidopsis miRNAs are described, many of which have differential expression patterns in development. Eight are absolutely conserved in the rice genome. The plant miRNA loci potentially encode stem-loop precursors similar to those processed by Dicer (a ribonuclease III) in animals. Mutation of an Arabidopsis Dicer homolog, CARPEL FACTORY, prevents the accumulation of miRNAs, showing that similar mechanisms direct miRNA processing in plants and animals. The previously described roles of CARPEL FACTORY in the development of Arabidopsis embryos, leaves, and floral meristems suggest that the miRNAs could play regulatory roles in the development of plants as well as animals.

Base Sequence↗
PubMed2006

Functional analysis of L-serine O-acetyltransferase from Corynebacterium glutamicum.

We report here the function of L-serine O-acetyltransferase (SAT) from the glutamic acid-producing bacterium Corynebacterium glutamicum. Based on the genome sequence of C. glutamicum and the NH(2)-terminal amino-acid sequence, the gene encoding SAT (cysE) was cloned and expressed in C. glutamicum. Deletion analysis of the 5'-noncoding region showed a putative -10 region ((-27)TTAAGT(-22) or (-26)TAAGTC(-21)) and a possible ribosome-binding site ((-12)AGA(-10)) just upstream from the start codon. We found that the SAT activity was sensitive to feedback inhibition by L-cysteine, and that SAT synthesis was repressed by L-methionine. Further, cysE-disrupted cells showed L-cysteine auxotrophy, indicating that C. glutamicum synthesizes L-cysteine from L-serine via O-acetyl-L-serine through the pathway involving SAT and O-acetyl-L-serine sulfhydrylase in the same manner as Escherichia coli.

Cloning, Molecular↗
PubMed2001

Hepatitis C virus genotyping based on 5' noncoding sequence analysis (Trugene).

Hepatitis C virus (HCV) genotyping of samples from 184 patients with chronic HCV infection by the Trugene 5'NC genotyping kit, based on sequence analysis of the 5' noncoding region (5' NCR), and the InnoLiPA assay was evaluated. In addition to these methods, the 184 samples were also analyzed by sequencing of part of the NS5B of the HCV genome after in-house PCR amplification, as a means of validating results obtained with the 5' NCR. The distribution of the genotypes typed by NS5B sequence analysis was as follows: 1a, 41 samples; 1b, 58 samples; 1d, 1 sample; 2a, 5 samples; 2b, 2 samples; 2c, 7 samples; 3a, 46 samples; 4a, 7 samples; 4c, 1 samples; 4e, 9 samples; 5a, 6 samples; 6a, 1 sample. The Trugene and InnoLiPA assays gave concordant results within HCV types in 100% of cases. The ability to discriminate at the subtype level was 76 and 74% for the Trugene and the InnoLiPA assays, respectively.

5' Untranslated Regions↗
PubMed1991

Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture.

Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant. RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells. RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells. Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro.

Animals↗
PubMed2003

RNA interference: biology, mechanism, and applications.

Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.

Animals↗
PubMed2002

Genomic structure and functional control of the Dlx3-7 bigene cluster.

The Dlx genes are involved in early vertebrate morphogenesis, notably of the head. The six Dlx genes of mammals are arranged in three convergently transcribed bigene clusters. In this study, we examine the regulation of the Dlx3-7 cluster of the mouse. We obtained and sequenced human and mouse P1 clones covering the entire Dlx3-7 cluster. Comparative analysis of the human and mouse sequences revealed several highly conserved noncoding regions within 30 kb of the Dlx3-7-coding regions. These conserved elements were located both 5' of the coding exons of each gene and in the intergenic region 3' of the exons, suggesting that some enhancers might be shared between genes. We also found that the protein sequence of Dlx7 is evolving more rapidly than that of Dlx3. We conducted a functional study of the 79-kb mouse genomic clone to locate cis-element activity able to reproduce the endogenous expression pattern by using transgenic mice. We inserted a lacZ reporter gene into the first exon of the Dlx3 gene by using homologous recombination in yeast. Strong lacZ expression in embryonic (E) stage E9.5 and E10.5 mouse embryos was found in the limb buds and first and second visceral arches, consistent with the endogenous Dlx3 expression pattern. This result shows that the 79-kb region contains the major cis-elements required to direct the endogenous expression of Dlx3 at stage E10.5. To test for enhancer location, we divided the construct in the mid-intergenic region and injected the Dlx3 gene portion. This shortened fragment lacking Dlx7-flanking sequences is able to drive expression in the limb buds but not in the visceral arches. This observation is consistent with a cis-regulatory enhancer-sharing model within the Dlx bigene cluster.

Animals↗
PubMed1982

Nucleotide sequence at the junction between the nonstructural and the structural genes of the semliki forest virus genome.

The nucleotide sequence at the junction between the nonstructural and the structural genes of the Semliki Forest virus 42S RNA genome has been determined from cloned cDNA. With the aid of S1-mapping, we have located the 5' end of the viral 26S RNA on this sequence. The 26S RNA is homologous to the 3' end of the 42S RNA and is used as a messenger for the structural proteins of the virus. The nucleotide sequence in the noncoding 5' region of the 26S RNA (51 bases) was thus established, completing the primary structure of the 26S RNA molecule (for earlier sequence work, see Garoff et al., Proc. Natl. Acad. Sci. U.S.A. 77:6376-6380, 1980, and Garoff et al., Nature (London) 288:236-241, 1980). An examination of the nucleotide sequences upstream from the initiator codon for the structural proteins on the 42S RNA genome shows that all reading frames are effectively blocked by stop codons, which means that the nonstructural genes in the 5' end of the 42S RNA molecule do not overlap with the structural ones at the 3' end of the molecule.

Base Sequence↗
PubMed2026

The chromatin reader ZMYND8 recruits the NuRD component GATAD2A through its MYND domain to regulate MAPT213 long noncoding RNA transcription.

The zinc finger MYND-type containing eight protein (ZMYND8) is a chromatin reader that regulates neuronal gene expression by controlling the microtubule-associated protein tau (MAPT) locus. Here, we investigate how ZMYND8 regulates expression of the long non-coding RNA MAPT213 through its interaction with GATA zinc finger domain containing 2A (GATAD2A), a component of the Nucleosome Remodelling and Deacetylase complex. ZMYND8 exhibits opposite regulatory effects on protein-coding MAPT and non-coding MAPT213 transcripts in a manner dependent on its MYND domain, promoting MAPT expression while suppressing MAPT213 levels. Chromatin immunoprecipitation experiments demonstrated that ZMYND8 specifically recruits GATAD2A to the MAPT213 internal regulatory region, establishing a direct link between protein binding and transcriptional control. We determined the crystal structure of the ZMYND8 coiled-coil MYND domain at high resolution, revealing a homodimeric architecture. The MYND domain specifically recognizes GATAD2A through direct interaction with proline-rich motifs in GATAD2A's central region. Structure-function analysis identified critical binding interface residues, while quantitative measurements revealed moderate-affinity interactions enhanced through multivalent binding mechanisms. These findings establish the molecular basis for ZMYND8-mediated recruitment of chromatin remodeling complexes to specific genomic loci and provide a structural framework for understanding transcriptional regulation of MAPT213.

Humans↗
PubMed2007

The DXPas34 repeat regulates random and imprinted X inactivation.

X chromosome inactivation (XCI) is initiated by expression of the noncoding Xist RNA in the female embryo. Tsix, the antisense noncoding partner of Xist, serves as its regulator during both imprinted and random XCI. Here, we show that Tsix in part acts through a 34mer repeat, DXPas34. DXPas34 contains bidirectional promoter activity, producing overlapping forward and reverse transcripts. We generate three new Tsix alleles in mouse embryonic stem cells and show that, while the Tsix promoter is unexpectedly dispensable, DXPas34 plays dual positive-negative functions. At the onset of XCI, DXPas34 stimulates Tsix expression through its enhancer activity. Once XCI is established, DXPas34 becomes repressive and stably silences Tsix. Germline transmission of the DXPas34 mutation demonstrates its necessity for both random and imprinted XCI in mice. Intriguingly, sequence analysis suggests that DXPas34 could potentially have descended from an ancient retrotransposon. We hypothesize that DXPas34 was acquired by Tsix to regulate antisense function.

Animals↗
PubMed2005

Folded RNA from an intron of one gene might inhibit expression of a counteracting gene.

Homeostatic maintenance of mRNA levels including prompt availability of mRNAs for translation in response to changing protein demands might be partly enabled by a system of combinatorial controls involving noncoding RNA blocking agents. This article proposes a specific version of that control mechanism, namely, a double-stranded RNA folding from transcription of an intron of one gene might and leading to an agent that inhibits mRNA of a counteracting gene. Thus transcription of the first gene would automatically repress translation of the second. On the basis of a bioinformatics search, we suggest a possible example, namely, that pro-apoptosis gene PAR4 might inhibit anti-apoptosis gene XIAP. Part of an intron from PAR4 folds to form a large, stable hairpin, and reverse complement of the hairpin stem (with approximately 280 nucleotides) matches a sub-sequence of an exon in XIAP. This would be part of an efficient system to drive initiated apoptosis to its conclusion. Figuratively speaking, it replaces two control knobs with one. Since repeats, some with many thousands of copies, occur throughout the genome with complex distributions, care must be taken before asserting that the presence of any repeat in any gene is significant. Our apoptosis example involves repeats, so experimental verification is needed and planned. However, if it is found that the noncoding RNA by-product of one gene folds into a hairpin that is processed into an agent that inhibits a counteracting gene, then the same type of control unit might be found extensively among counteracting families of genes of many types.

Animals↗
PubMed2005

Structure and transcription promoter activity of mouse flap endonuclease 1 gene: alternative splicing and bidirectional promoter.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease involved in DNA replication and repair. The mouse Fen1 gene, which has two exons, is located immediately adjacent to the gene corresponding to full-length cDNA of Riken 1810006K21 (1810006K21Rik) in a head-to-head orientation. Transcription initiation sites of each gene are 274 bp apart in the mouse genome. The spacer sequence between the bidirectional genes contains a CpG island, but lacks the typical TATA box. In the present study, transcription of the mFen1 gene was started from two initiation sites, and the first noncoding exon was spliced to the second exon using two different splicing donor sites, producing 3 kinds of mFen1 transcripts. A 594-bp fragment between the mFen1 and 1810006K21Rik genes, which contains two conserved sequence blocks (CSB) between mouse and human sequence, functions as a bidirectional promoter. The multiple cis-elements, including an Ets-binding site and E-box in the CSB, are involved in activation or repression of transcription in both directions. Interestingly, the E-box activates mFen1 transcription and simultaneously represses promoter activity in the opposite direction. Mutation of either splicing donor site of the mFen1 gene produced limiting alternative splicing products, but did not affect luciferase activity. In contrast, the splicing-defective mutation produced by disruption of the acceptor site completely lacked luciferase activity, indicating that the splicing has a significant effect on production of luciferase protein by shortening the 5'-untranslated region of the mRNA.

5' Untranslated Regions↗
PubMed2006

Constitutional UPD for chromosome 11p15 in individuals with isolated hemihyperplasia is associated with high tumor risk and occurs following assisted reproductive technologies.

Isolated hemihyperplasia (IH) refers to a distinct diagnosis involving asymmetric overgrowth of single or multiple organs or regions of the body and can result from various genomic changes including molecular alterations of 11p15; these are paternal uniparental disomy (UPD), and alterations of methylation at two imprinting centers at 11p15: IC1 (H19) and IC2 (KCNQ1OT1). As little information is available on the molecular basis of tumor development in IH, or on the frequency of tumors in children with different molecular subtypes of IH, molecular testing was undertaken on 51 patients with IH and revealed: 8 (16%) with UPD, 3 (6%) with hypomethylation at KCNQ1OT1, and 0 with hypermethylation at H19. Of the 8 patients with UPD, 4 had tumors (3 hepatoblastomas, 1 Wilms tumor); 0/3 patients with hypomethylation at KCNQ1OT1 had a tumor; of the remaining 40 with no molecular alterations, 6 had tumors (3 Wilms tumors, 2 neuroblastomas, 1 adrenocortical adenoma). The 50% tumor frequency in patients with IH and UPD was statistically significantly higher than the 15% tumor frequency in those with IH and no molecular alteration detected (Fisher's exact test P = 0.047, OR 5.67). This is the first demonstration that UPD at 11p15 in patients with IH confers a higher tumor risk than in patients with IH without this molecular change. Of note, two of the eight patients with UPD and IH were conceived using assisted reproductive technologies (ART), thus raising the question whether ART might impact the rate of somatic recombination during embryonic development.

Abnormalities, Multiple↗
PubMed1995

Expression of X-linked genes in androgenetic, gynogenetic, and normal mouse preimplantation embryos.

A quantitative RT-PCR approach has been used to examine the expression of a number of X-linked genes during preimplantation development of normal mouse embryos and in androgenetic and gynogenetic mouse embryos. The data reveal moderately reduced expression of the Prps1, Hprt, and Pdha1 mRNAs in androgenetic eight-cell and morula stage embryos, but not in androgenetic blastocysts. Pgk1 mRNA abundance was severely reduced in androgenones at the eight-cell and morula stages and remained reduced, but to a lesser degree, in androgenetic blastocysts. These data indicate that paternally inherited X chromosomes are at least partially repressed in androgenones, as they are in normal XX embryos, and that the degree of this repression is chromosome position-dependent or gene-dependent. Gynogenetic embryos expressed elevated amounts of some mRNAs at the morula and blastocyst stages, indicative of a delay in dosage compensation that may be chromosome position-dependent. The Xist RNA was expressed at a greater abundance in androgenones than in gynogenones at the eight-cell and morula stages, consistent with previous studies. Xist expression was observed in both androgenones and gynogenones at the blastocyst stage. We conclude that the developmental arrest in early androgenones may be, in part, due to reduced expression of essential X-linked genes, particularly those near the X inactivation center, whereas the developmental defects of gynogenones and parthenogenones, by contrast, may be partially due to overexpression of X-linked genes in extraembryonic tissues, possibly those farthest away from the X inactivation center.

Animals↗
PubMed1999

Monoallelic expression of reactivated imprinted genes in embryonal carcinoma cell hybrids.

Though DNA methylation is necessary to maintain monoallelic expression of imprinted genes, it is still unclear whether it represents the primary mark. Here we ask whether the imprinting mark is still present in terminally differentiated somatic cells in which the transcription of embryo-specific imprinted genes was shut off. For such analysis H19 and Igf2 genes were activated by inducing differentiation of (mouse embryonal carcinoma cell x mouse lymphocyte) hybrid cell clones. Although lymphocytes do not express H19 and Igf2, both genes are reactivated in a proper monoallelic manner in hybrid cells. Analysis of the upstream region of the H19 gene confirmed maintenance of differential methylation of the active and inactive H19 genes of lymphocyte origin, although a tendency toward in vitro induced hypermethylation was apparent. We conclude that the imprints of the H19, U2af1-rs1, and Igf2 genes are maintained in lymphocytes in adult mice.

Alleles↗
PubMed1998

Bisulfite sequencing in preimplantation embryos: DNA methylation profile of the upstream region of the mouse imprinted H19 gene.

In this study we describe a modification of the bisulfite genomic sequencing protocol that enables detection of methylation from as few as five diploid cells from preimplantation mouse embryos. We have used bisulfite genomic sequencing to study the methylation profile of the putative imprinting element upstream of the mouse H19 gene at several stages of embryonic development, including fertilized oocytes and two-cell embryos. The methylation of the H19 imprinting element has recently been described extensively for midgestation embryos, but remains poorly characterized for the preimplantation stages of development, despite widespread changes in genomic DNA methylation occurring at this time. We studied the methylation profile of 35 CpG sites spanning two regions within the H19 imprinting element and found that an overall pattern of allele-specific methylation was maintained at all developmental stages examined, including fertilized oocytes and two-cell embryos. However, allele-specific methylation was not maintained in an absolute fashion subsequent to the first cell division, with a clear flux between partial de novo methylation of the maternal allele and partial demethylation of the paternal allele. Our findings highlight the dynamics of methylation in the early embryo and suggest that it is the overall level of methylation that is responsible for maintenance of the imprinting element and not the methylation of individual CpG sites.

Alleles↗