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At least 1,567 records · Page 87Linked to original sources
PubMed1998

Characterization of novel parent-specific epigenetic modifications upstream of the imprinted mouse H19 gene.

Genomic imprinting results in parent-specific monoallelic expression of a small number of genes in mammals. The identity of imprints is unknown, but much evidence points to a role for DNA methylation. The maternal alleles of the imprinted H19 gene are active and hypomethylated; the paternal alleles are inactive and hypermethylated. Roles for other epigenetic modifications are suggested by allele-specific differences in nuclease hypersensitivity at particular sites. To further analyze the possible epigenetic mechanisms determining monoallelic expression of H19, we have conducted in vivo dimethylsulfate and DNase I footprinting of regions upstream of the coding sequence in parthenogenetic and androgenetic embryonic stem cells. These cells carry only maternally and paternally derived alleles, respectively. We observed the presence of maternal-allele-specific dimethylsulfate and DNase I footprints at the promoter indicative of protein-DNA interactions at a CCAAT box and at binding sites for transcription factors Sp1 and AP-2. Also, at the boundary of a region further upstream for which existent differential methylation has been suggested to constitute an imprint, we observed a number of strand-specific dimethylsulfate reactivity differences specific to the maternal allele, along with an unusual chromatin structure in that both strands of maternally derived DNA were strongly hypersensitive to DNase I cutting over a distance of 100 nucleotides. We therefore reveal the existence of novel parent-specific epigenetic modifications, which in addition to DNA methylation, could constitute imprints or maintain monoallelic expression of H19.

Alleles↗
PubMed2006

Establishment and maintenance of H19 imprinting in the germline and preimplantation embryo.

The mouse H19 and Igf2 genes are oppositely imprinted and share enhancers that reside 3' to the genes. The imprinted expression of these genes is coordinated by a 2-kb regulatory element, the differentially methylated domain (DMD), positioned between the two genes. The methylation status of this region determines the ability of the insulator factor CTCF to bind to its sites in the DMD. Deletions and mutations of the DMD that affect imprinting in the soma have little effect on the methylation pattern of H19 in the germline, suggesting that additional sequences and factors contribute to the earliest stages of imprinting regulation at this locus. Less is known about these initial steps, which include the marking of the parental alleles, the onset of allele-specific expression patterns and maintenance of the imprints in the preimplantation embryo. Here, we will focus on these early steps, summarizing what is known and what questions remain to be addressed.

Animals↗
PubMed2004

Identification of microRNAs and other tiny noncoding RNAs by cDNA cloning.

MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the approximately 22-nt fraction of total RNA. Methods are described for the construction of cDNA libraries from small noncoding RNAs through the use of T4 RNA ligase, reverse transcriptase, and polymerase chain reaction. cDNAs are cloned in lambda or plasmid vectors, and the sequences are compared to annotated genomic sequence databases, and analyzed by RNA folding programs to distinguish miRNA sequences from other small RNAs of similar size. Northern blot hybridization is used to confirm the expression of small RNAs in vivo.

3' Untranslated Regions↗
PubMed2001

Evidence that insulin is imprinted in the human yolk sac.

Allelic variation in the size of the insulin (INS) variable number tandem repeat (VNTR) correlates with the expression of both INS in the pancreas and thymus and IGF2 (the gene downstream of INS) in the placenta. In addition, the shorter, class I alleles are associated with type 1 diabetes, whereas the longer, class III alleles are associated with type 2 diabetes, polycystic ovary syndrome (PCOS), and size at birth. Parent-of-origin effects have been reported for type 2 diabetes and PCOS, thus implicating a role for genomic imprinting in these phenotypes. In mice, Ins2 is imprinted and paternally expressed in the yolk sac. In humans, evidence for the imprinting of INS is circumstantial, with occasional monoallelic expression in the thymus. In the present study, we found evidence for the imprinted paternal expression of INS in the human yolk sac. Two other imprinted genes from the same cluster are also expressed monoallelically in the human yolk sac. IGF2 was expressed solely from the paternal allele, and H19 was expressed solely from the maternal allele. These data suggest not only further functional roles for the human yolk sac in early fetal growth, but also evidence for a potential causal link between the control of insulin expression during development and insulin/growth-related diseases in later life.

Alleles↗
PubMed2001

Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat.

Sequence databases could be efficiently exploited for development of DNA markers if it were known which gene regions reveal the most polymorphism when amplified by PCR. We developed PCR primer pairs that target specific regions of previously sequenced genes from Avena and Zea species. Primers were targeted to amplify 40 introns, 24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48 maize inbred lines (previously assayed for simple-sequence repeat (SSR) polymorphism) for amplification-product polymorphism. We also developed primers to target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22 hexaploid oat cultivars and 2 diploid Avena species for amplification-product polymorphism. In maize, 67% of promoter markers, 58% of intron markers, and 13% of exon markers exhibited amplification-product polymorphisms. Among polymorphic primer pairs in maize, genotype diversity was highest for SSR markers (0.60) followed by intron markers (0.46), exon markers (0.42), and promoter markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58% of intron markers revealed polymorphisms, but among the cultivars only, 21% of SSR markers and 50% of intron markers were polymorphic. Polymorphic-sequence-tagged sites for plant-breeding applications can be created easily by targeting noncoding gene regions.

Avena↗
PubMed1992

Human alpha 2(VI) collagen gene. Heterogeneity at the 5'-untranslated region generated by an alternate exon.

Cosmid clones containing the 5' region of the human alpha 2(VI) collagen gene have been isolated and characterized. DNA sequencing indicates that the signal peptide and the amino-globular domain are encoded by four exons of 142, 596, 21, and 66 base pairs (bp). However, S1 nuclease and primer extension analyses show that the transcription start site is not present in the 142-bp exon. Two different 5' cDNA clones are generated by the anchored polymerase chain reaction. Using the 5' cDNA clones as probes, two untranslated exons (1, 1A) are found 12 kilobase pairs upstream of the first coding exon. These two exons are alternatively used in human fibroblasts, and most transcripts contain exon 1 sequence. Exon 1 shows, by primer extension and S1 nuclease protection assay, two major and several minor transcription start sites. The promoter region contains a canonical TATA box, seven GGGCGG sequences, two possible CAAT boxes, and two sequences resembling AP2 binding sites. Exon 1A contains three alternative splice donor sites and is located 650 bp downstream of exon 1. The most 3' splice donor site of exon 1A is found within an Alu repeat sequence. Exon 1A is preceded by five GGGCGG sequences and one resembling the AP2 binding site although neither TATA or CAAT boxes are found. Two additional GGGCGG sequences are located at the beginning of exon 1A. This study establishes that the human alpha 2(VI) collagen gene is 36 kilobase pairs long and contains 30 exons. The 5'-untranslated and promoter regions are significantly different from the corresponding segments of the chicken gene. The human gene produces by alternative processing multiple mRNAs differing in the 5'-untranslated region as well as the 3'-coding and noncoding sequences.

Amino Acid Sequence↗
PubMed1985

A gene encoding rat cholecystokinin. Isolation, nucleotide sequence, and promoter activity.

The gene for rat cholecystokinin (CCK) was isolated from a rat genomic DNA library. The transcription unit spans 7 kilobases and is interrupted by two introns. The initiator methionine codon lies 2 bases into exon 2; therefore, exon 1 is a noncoding exon. The transcription initiation site was determined using avian myeloblastosis reverse transcriptase, a cDNA primer, and mRNA isolated from a rat medullary thyroid carcinoma. A "TATA"-like sequence precedes the transcription initiation site at position -34. The polyadenylation site for the gene was mapped by a nuclease protection assay using a cRNA generated by transcription of the exon 3 region of the CCK gene with SP6 bacteriophage RNA polymerase. The sequence AT-TAAA is found 22 bases 5' to the site determined to be the polyadenylation addition site. Two regions of simple repetitive DNA occur within the CCK lambda clone, one within intron 2 and the other 4 kilobases 3' to the gene. Sequence analysis of the repetitive element 3' distal to the gene revealed two copies of the sequence 5'-(AC)n-3', where n is 22 and 25. A 114-base pair sequence of predominantly repeating purine-pyrimidine nucleotides separates these two d(AC) repeats. Transcriptional control elements were investigated by fusing regions of the CCK gene to the structural gene encoding chloramphenicol acetyltransferase. Promoter activity was determined by transfecting COS-7 cells with plasmids containing the gene fusions, followed by determining chloramphenicol acetyltransferase activity in cellular extracts. The region necessary for expression of the CCK gene fusions in COS-7 cells is within 144 bases 5' to the initiation of transcription.

Animals↗
PubMed2006

Candidate single nucleotide polymorphism selection using publicly available tools: a guide for epidemiologists.

Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation, with millions present in the human genome. Because only 1% might be expected to confer more than modest individual effects in association studies, the selection of predictive candidate variants for complex disease analyses is formidable. Technologic advances in SNP discovery and the ever-changing annotation of the genome have led to massive informational resources that can be difficult to master across disciplines. A simplified guide is needed. Although methods for evaluating nonsynonymous coding SNPs are known, several other publicly available computational tools can be utilized to assess polymorphic variants in noncoding regions. As an example, the authors applied multiple methods to select SNPs in DNA double-strand break repair genes. They chose to evaluate SNPs that occurred among a preexisting set of 57 validated assays and to justify new assay development for 83 potential SNPs in the DNA-dependent protein kinase catalytic subunit. Of the 140 SNPs, the authors eliminated 119 variants with low or neutral predictions. The existing computational methods they used and the semiquantitative relative ranking strategy they developed can be adapted to a priori SNP selection or post hoc evaluation of variants identified in whole genome scans or within haplotype blocks associated with disease. The authors show a "real world" application of some existing bioinformatics tools for use in large epidemiologic studies and genetic analyses. They also reviewed alternative approaches that provide related information.

Algorithms↗
PubMed2004

Discovery of functional noncoding elements by digital analysis of chromatin structure.

We developed a quantitative methodology, digital analysis of chromatin structure (DACS), for high-throughput, automated mapping of DNase I-hypersensitive sites and associated cis-regulatory sequences in the human and other complex genomes. We used 19/20-bp genomic DNA tags to localize individual DNase I cutting events in nuclear chromatin and produced approximately 257,000 tags from erythroid cells. Tags were mapped to the human genome, and a quantitative algorithm was applied to discriminate statistically significant clusters of independent DNase I cutting events. We show that such clusters identify both known regulatory sequences and previously unrecognized functional elements across the genome. We used in silico simulation to demonstrate that DACS is capable of efficient and accurate localization of the majority of DNase I-hypersensitive sites in the human genome without requiring an independent validation step. A unique feature of DACS is that it permits unbiased evaluation of the chromatin state of regulatory sequences from widely separated genomic loci. We found surprisingly large differences in the accessibility of distant regulatory sequences, suggesting the existence of a hierarchy of nuclear organization that escapes detection by conventional chromatin assays.

Chromatin↗
PubMed2026

Germline noncoding risk variants influence clonal hematopoiesis through altered hematopoietic enhancer activity.

Clonal hematopoiesis of indeterminate potential (CHIP) is a precursor condition characterized by the expansion of mutant hematopoietic stem and progenitor cell (HSPC) clones that increases the risk of hematologic malignancies. Although genome-wide association studies have identified multiple non-coding loci associated with CHIP susceptibility, their mechanisms remain unclear. We hypothesized that CHIP risk variants alter enhancer activity in HSPCs. To test this, we screened 1,374 non-coding variants from 51 CHIP-associated loci using a Massively Parallel Reporter Assay (MPRA) in the CD34+ fraction of MUTZ-3 cells. We identified 87 regulatory variants across 32 loci. Targeted genome editing in hematopoietic cells and complementary reporter assays in primary human HSPCs validated enhancer activity for variants regulating NKD2, FLT3, and MSI2. Functional studies demonstrated that increased MSI2 expression, modeling the effect of the CHIP risk allele, promotes clonal expansion of TET2-deficient HSPCs, providing a mechanistic link between inherited non-coding variation and CHIP clonal expansion.

Journal Article↗
PubMed1998

Identification of a novel bone morphogenetic protein-responsive gene that may function as a noncoding RNA.

Bone morphogenetic proteins (BMPs)/osteogenic proteins (OPs), members of the transforming growth factor-beta superfamily, have a wide variety of effects on many cell types including osteoblasts and chondroblasts, and play critical roles in embryonic development. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. Signaling by these receptors is mediated by the recently identified Smad proteins. Despite the rapid progress in understanding of the signaling mechanism downstream of BMP receptors, the target genes of BMPs are poorly understood in mammals. Here we identified a novel gene, termed BMP/OP-responsive gene (BORG), in C2C12 mouse myoblast cell line which trans-differentiates into osteoblastic cells in response to BMPs. Expression of BORG was dramatically induced in C2C12 cells by the treatment with BMP-2 or OP-1 within 2 h and peaked at 12-24 h, whereas transforming growth factor-beta had a minimal effect. BMP-dependent expression of BORG was also detected in other cell types which are known to respond to BMPs, suggesting that BORG is a common target gene of BMPs. Cloning and sequence analysis of BORG cDNA and the genomic clones revealed that, unexpectedly, the transcript of BORG lacks any extensive open reading frames and contains a cluster of multiple interspersed repetitive sequences in its middle part. The unusual structural features suggested that BORG may function as a noncoding RNA, although it is spliced and polyadenylated as authentic protein-coding mRNAs. Together with the observation that transfection of antisense oligonucleotides of BORG partially inhibited BMP-induced differentiation in C2C12 cells, it is possible that a new class of RNA molecules may have certain roles in the differentiation process induced by BMPs.

Animals↗
PubMed2005

Evidence for widespread degradation of gene control regions in hominid genomes.

Although sequences containing regulatory elements located close to protein-coding genes are often only weakly conserved during evolution, comparisons of rodent genomes have implied that these sequences are subject to some selective constraints. Evolutionary conservation is particularly apparent upstream of coding sequences and in first introns, regions that are enriched for regulatory elements. By comparing the human and chimpanzee genomes, we show here that there is almost no evidence for conservation in these regions in hominids. Furthermore, we show that gene expression is diverging more rapidly in hominids than in murids per unit of neutral sequence divergence. By combining data on polymorphism levels in human noncoding DNA and the corresponding human-chimpanzee divergence, we show that the proportion of adaptive substitutions in these regions in hominids is very low. It therefore seems likely that the lack of conservation and increased rate of gene expression divergence are caused by a reduction in the effectiveness of natural selection against deleterious mutations because of the low effective population sizes of hominids. This has resulted in the accumulation of a large number of deleterious mutations in sequences containing gene control elements and hence a widespread degradation of the genome during the evolution of humans and chimpanzees.

Animals↗
PubMed1998

[Exon-intron structure of the fet5+ gene of Schizosaccharomyces pombe and physical mapping of genome encompassing regions].

Plasmid pYUK3 bearing the fet5+ gene of Schizosaccharomyces pombe was isolated from a genomic library of the fission yeast, and a detailed physical map of the whole genomic insert (ca. 9.6 Kbp) was constructed. The primary structure of the fet5+ gene and its flanking regions is established. The gene contains a single 45-bp intron in its distal part. A typical TATA-box (TATAAG) was found in the 5'-noncoding region ca. 50 bp upstream of the putative start of transcription, and the 3'-noncoding region contains AT-rich palindromes, which are probably involved in termination of the fet5+ transcription. A previously unidentified gene of Sz. pombe encoding a protein with some similarity to one of the transcriptional activators from the TBP (TATA-binding protein) group of SPT factors of transcription was found in the vicinity of the fet5+ gene. Taking into account that cDNA of the fet5(+)-gene was isolated as a suppressor of the genetic-defect of nuclear RNA polymerases I-III (Bioorg. Khim., 1997, vol. 23, No 3, pp. 234-237), this vicinity may be the first evidence of possible clustering, in the genome of the fission yeast, of genes participating in transcription regulation.

Alternative Splicing↗
PubMed1998

Molecular and in vitro analysis of eight dengue type 2 viruses isolated from patients exhibiting different disease severities.

Potential genetic determinants of dengue virulence were studied by sequencing the entire genomes of eight dengue 2 virus strains isolated from patients exhibiting different disease severities during an epidemic season in northeastern Thailand in 1993. The isolates came from one dengue shock syndrome (ThNH-7/93), three dengue hemorrhagic fever, and four dengue fever patients. Phylogenetic analysis showed that the isolates belonged to the Southeast Asian genotype. The 3' noncoding regions showed distinctive secondary structures, with one specific structure for the isolate ThNH-7/93. Analysis of the predicted polyprotein showed several amino acid (aa) changes scattered mostly in the nonstructural region. Of 30 positions with aa changes, 7 were unique to the isolate ThNH-7/93 and 3 of those led to radical alterations in aa character. Several aa changes coincided with previous studies relating genome sequence and virulence. Minimal changes in computer-predicted protein secondary structures were observed. Infective particles in the inoculum for all isolates were approximately equal as measured by focus formation on BHK-21 cells, but this did not correlate with the number of plaques formed on LLC-MK2 cells. Isolates from patients that experienced secondary infection were shown to have significantly larger plaques than the isolates from primary infection patients.

Adolescent↗
PubMed1994

[Single-primer variant of PCR-amplifications of segments of the main noncoding region of human mitochondrial DNA].

A method for amplifying segments of the main non-coding region of human mitochondrial DNA using a single-primer PCR is proposed. The method employs oligonucleotides that are homologous to this region of the human mitochondrial genome. Possible reaction mechanisms and the perspectives for using the method in evolutionary and population genetic studies are discussed.

Base Sequence↗
PubMedDate not supplied

A novel human type I hair keratin gene: evidence for two keratin hHa3 isoforms.

We present the nucleotide and amino acid sequence for a novel human type I hair keratin, which could be identified through its high sequence homology and strict carboxyterminal length identity as a human ortholog of the murine hair keratin mHa3. Our hHa3 sequence differs, however, from that of a previously described hHa3 hair keratin (published only as an amino acid sequence; [13]) in 24 amino acid position, 8 of which occur in the middle of the carboxyterminal domain. PCR of genomic DNA from 25 normal human subjects using a primer pair derived from sequence segments located in the 3'-region of our hHa3 clone that encode conserved amino acid sequences in both keratins, resulted in the amplification of two distinct products of 0.38 kbp and 1.0 kbp. DNA sequence analysis of the cloned PCR products allowed identification of the 0.38 kb sequence as that originating from Yu et al. [13] and the 1.0 kb sequence as that being derived from our data. The difference in fragment length was due to unique intron 6 sequences, indicating that these two keratin species are encoded by genes of their own. Moreover, extensive Southern blot analyses with DNA from 25 unrelated individuals of different races using a 3'-noncoding sequence from our keratin and the intron 6 sequence of the keratin of Yu et al. [13], as hybridization probes showed that both keratin genes are present as single copy sequences occurring ubiquitously and without gross alterations in the human genome. Collectively, these data demonstrate that the human type I hair keratin described in this paper represents an isoform of the previously described hHa3 keratin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Synthesis of infectious in vitro transcripts from Cassia yellow blotch bromovirus cDNA clones and a reassortment analysis with other bromoviruses in protoplasts.

Cassia yellow blotch virus (CYBV), genus Bromovirus, was isolated from the Australian native legume, Cassia pleurocarpa, in western Queensland, and its host range was found to be distinct from other bromoviruses. In this study, CYBV was shown to infect systemically and efficiently a model plant species, Arabidopsis thaliana, as we recently reported for another bromovirus, Spring beauty latent virus (SBLV). We constructed full-length cDNA clones of CYBV genomic RNAs from which infectious in vitro transcripts can be transcribed, and determined their complete nucleotide sequences. CYBV RNA3 contains the box B motif in the intercistronic region, but lacks the subgenomic promoter-like sequence in the 5' noncoding region, as does Brome mosaic virus (BMV). To understand relationships among bromoviruses, we generated reassortants between CYBV and three other bromoviruses, BMV, SBLV and Cowpea chlorotic mottle virus. We found that all reassortants between BMV and CYBV accumulated viral RNAs to detectable levels in protoplasts of Nicotiana benthamiana, even when RNAs 1 and 2, which encode the replication proteins 1a and 2a, respectively, were heterologous. Sequence comparison and reassortment experiments of CYBV and other bromoviruses demonstrated that CYBV is closely related to BMV.

Bromovirus↗
PubMed2004

Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries.

Comprehensive identification of sequences that regulate transcription is one of the major goals of genome biology. Focal alteration in chromatin structure in vivo, detectable through hypersensitivity to DNaseI and other nucleases, is the sine qua non of a diverse cast of transcriptional regulatory elements including enhancers, promoters, insulators, and locus control regions. We developed an approach for genome-scale identification of DNaseI hypersensitive sites (HSs) via isolation and cloning of in vivo DNaseI cleavage sites to create libraries of active chromatin sequences (ACSs). Here, we describe analysis of >61,000 ACSs derived from erythroid cells. We observed peaks in the density of ACSs at the transcriptional start sites of known genes at non-gene-associated CpG islands, and, to a lesser degree, at evolutionarily conserved noncoding sequences. Peaks in ACS density paralleled the distribution of DNaseI HSs. ACSs and DNaseI HSs were distributed between both expressed and nonexpressed genes, suggesting that a large proportion of genes reside within open chromatin domains. The results permit a quantitative approximation of the distribution of HSs and classical cis-regulatory sequences in the human genome.

Chromatin↗