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At least 1,423 records · Page 79Linked to original sources
PubMed1997

RIRE1, a retrotransposon from wild rice Oryza australiensis.

RIRE1 is a retrotransposon present in wild rice Oryza australiensis in an extraordinary number of copies, and only a portion of the LTR sequence has been determined previously. Here, we isolated and sequenced DNA segments of various portions of RIRE1, revealing that the sequences of LTR and the internal region were 1523 and 5277 bp in length, respectively. The internal region shows homology with the pol region in copia, a Drosophila retrotransposon, indicating that RIRE1 is a copia-like retrotransposon. The internal region of RIRE1 contained an open reading frame coding for genes, gag, pro, int, rt and rh, like copia and retroelements related to it. A clone screened from a library of the O. australiensis genomic DNA contained solo LTR, which was flanked by direct repeats of a 5-bp sequence. This suggests that RIRE1 generates a duplication of the target sequence of 5 bp upon retroposition. We observed that many RIRE1 members were nested by another RIRE1 member. This indicates that these RIRE1 members have received another RIRE1 to make an extraordinary number of copies in the O. australiensis genome without giving a deleterious effect on the growth of rice cells.

Amino Acid Sequence↗
PubMed2004

In situ hybridization: detecting viral nucleic acid in formalin-fixed, paraffin-embedded tissue samples.

In situ hybridization is a method for detecting specific nucleic acid sequences within individual cells. This technique permits visualization of viral nucleic acid or gene expression in individual cells within their histologic context. In situ hybridization is based on the complementary binding of a labeled nucleic acid probe to complementary sequences in cells or tissue sections, followed by visualization of target sequences within the cells. It has been used widely for the detection of viral nucleic acid sequences within individual cells. This review will define the technical approaches of in situ hybridization and its current application to detect viral nucleic acids within formalin-fixed, paraffin-embedded tissue samples, with special reference to the Epstein-Barr virus.

DNA, Viral↗
PubMed1997

Retrieval of DNA sequences present at an extremely low frequency.

We describe a method for retrieving sequences with one or two point mutations of a given target sequence, which are present in a DNA population at a frequency of 1 in 466 x 10(3) and 1 in 28 x 10(3) molecules, respectively. By stringent hybridization to a stable, chemically immobilized probe, a large excess of unrelated fragments is removed, and the bound sequences are dissociated and amplified. By repeating the hybridization-amplification cycles twice, we achieved an estimated enrichment of 404,000-fold and 1612-fold, respectively, which was confirmed by cloning the resultant products and sequencing 35 clones. This procedure can be applied to retrieve mutated sequences that exist at an extremely low frequency in a DNA population.

Cloning, Molecular↗
PubMed1994

The role of mitotic recombination in carcinogenesis.

Genetic recombination systems are present in all living cells and viruses and generally contribute to their hosts' flexibility with respect to changing environmental conditions. Recombination systems not only help highly developed organisms to protect themselves from microbial attack via an elaborate immune system, but conversely, recombination systems also enable microorganisms to escape from such an immune system. Recombination enzymes act with a high specificity on DNA sequences that either exhibit extended stretches of homology or contain characteristic signal sequences. However, recombination enzymes may rarely act on incorrect alternative target sequences, which may result in the formation of chromosomal deletions, inversions, translocations, or amplifications of defined DNA regions. This review describes the characteristics of several recombination systems and focuses on the implication of aberrant recombination in carcinogenesis. The consequences of mitotic recombination on the inappropriate activation of protooncogenes and on the loss of tumor suppressor genes is discussed. Cases are reported where mitotic recombination clearly has been associated with carcinogenesis in rodents as well as humans. Several test systems able to detect recombinagenic activities of chemical compounds are described.

Animals↗
PubMed1994

Functional cytochrome P4503A isoforms in human embryonic tissues: expression during organogenesis.

Expression of functional cytochrome P450 (CYP) isoforms in human embryonic tissues was explored during organogenesis (days 50-60 of gestation) with substrate probes, inhibitor probes, and immunoprobes and by reverse transcription-polymerase chain reaction (PCR), cloning, and sequencing. Evidence was obtained for the presence of relatively high levels of one or more functional CYP3A isoforms in embryonic livers. This was manifested as relatively extensive hydroxylation of (R)-warfarin at carbon 10 and as triacetyloleandomycin-inhibited O-debenzylation of benzyloxyresorufin when human embryonic hepatic microsomal fractions were used as enzyme sources. Immunoblots with anti-CYP3A4 antibody exhibited a strong signal in embryonic hepatic tissues but, in contrast, indicated very low or negligible CYP3A levels in human embryonic lung, kidney, heart, adrenal, and brain tissues. To explore expression of individual members of the CYP3A subfamily in human embryonic hepatic tissues at this early gestational stage, CYP3A cDNA was generated by reverse transcription, amplified by PCR, cloned, and sequenced. Oligonucleotide primers used for PCR were designed to flank target sequences unique to CYP3A but also common to all human CYP3A subfamily members for which GenBank nucleotide sequence information was available (CYP3A3, CYP3A4, CYP3A5, CYP3A5P, and CYP3A7). Sequencing data indicated that plasmids in 58 of 59 recombinant positive colonies contained an insert with a sequence identical to that present in CYP3A7 cDNA and the plasmid of only one colony contained an insert with a sequence identical to that present in CYP3A5 cDNA. No evidence was found for expression of CYP3A3 or CYP3A4. Thus, during organogenesis, human embryonic hepatic tissues express primarily CYP3A7 and are capable of significant CYP3A7-catalyzed xenobiotic monooxygenation during this very early stage of gestation.

Base Sequence↗
PubMed2001

A gene required for the novel activation of a class II DNA photolyase in Chlamydomonas.

DNA photolyases catalyze the blue light-dependent repair of UV light-induced damage in DNA. DNA photolyases are specific for either cyclobutane-type pyrimidine dimers or (6-4) photoproducts. PHR2 is a gene that in Chlamydomonas reinhardtii encodes a class II DNA photolyase which catalyzes the photorepair of cyclobutane-type pyrimidine dimers. Based on amino acid sequence analysis of PHR2, which indicates the presence of a chloroplast targeting sequence, PHR2 was predicted to encode the chloroplast photolyase of Chlamydomonas. Using a sensitive gene-specific in vivo repair assay, we found that overexpression of PHR2 in Chlamydomonas results in targeting of the protein to not only the chloroplast, but also to the nucleus. Overexpression of PHR2 photolyase in a photoreactivation-deficient mutant, phr1, results in a largely inactive product. The phr1 mutant was found to be deficient in both photorepair of a chloroplast gene, rbcL, and a nuclear gene, rDNA. These results suggest that PHR2 is the structural gene for the photolyase targeted to both the chloroplast and the nucleus, and that the PHR1 gene product is necessary for full activity of PHR2 protein. To our knowledge, the requirement for a second gene for full activity of a DNA photolyase is novel.

Animals↗
PubMed2005

siRNA targeting the leader sequence of SARS-CoV inhibits virus replication.

SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.

5' Untranslated Regions↗
PubMed2004

Differential enzyme targeting as an evolutionary adaptation to herbivory in carnivora.

Not all members of the order Carnivora are carnivorous. Some are omnivorous, and a few, such as the giant panda, Ailuropoda melanoleuca, are almost exclusively herbivorous. Although a number of adaptations to increased plant-eating are recognized within Carnivora, few have been studied at the molecular level. One molecular adaptation to diet that is spread widely across Mammalia is the differential intracellular targeting of the intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT), which tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. In the present study, we have analyzed the targeting of AGT in Carnivora in relation to species' natural diets. We show not only that there has been an adaptive shift in AGT targeting from the mitochondrion toward the peroxisome as diets have shifted from being mainly carnivorous to ones that are more omnivorous and herbivorous but also that in one lineage, namely that of the giant panda, there is evidence for positive selection pressure at the molecular level on the AGT mitochondrial targeting sequence to decrease its efficiency, thereby allowing more AGT to be targeted to the peroxisomes.

Adaptation, Biological↗
PubMed1993

The Saccharomyces cerevisiae MTS1 gene encodes a putative RNA-binding protein involved in mitochondrial protein targeting.

Most proteins present in the mitochondrion are nuclear encoded, and are directed to the organelle by virtue of a targeting sequence at the N terminus of the precursor protein. Mitochondrial (mt) protein targeting appears to require several accessory proteins that recognise mt precursors both in the cytoplasm and at the mt surface. We describe here the use of yeast genetics to identify a protein that is required for mt protein targeting. Two yeast mutants (mts1 and mts2) were isolated as extragenic suppressors of a known targeting defect in the presequence of the beta-subunit of ATP synthase. We have cloned and sequenced the wild-type allele of one of these genes (MTS1) and shown that it encodes a member of a family of RNA-binding proteins that is essential for growth.

Amino Acid Sequence↗
PubMed1988

Highly preferred targets for retrovirus integration.

A central feature of retrovirus replication is integration of the provirus into host cell DNA, but the specificity of this step for cell target sequences has not been clarified. To investigate this issue, we developed a method for screening and comparing large numbers of unselected integration events. Using a replication-competent Rous sarcoma virus containing a bacterial suppressor tRNA gene as a selectable marker, we obtained collections of clones comprising integrated provirus together with host flanking sequences. Hybridization and sequence analysis of the flanking sequence reveals the presence of a number of strongly preferred integration targets. Within these targets, independent integration events occur at sites identical to the base.

Animals↗
PubMed2006

Characterization of membrane association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein.

Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.

Amino Acid Sequence↗
PubMed2001

[Real-Time Quantitative PCR and Detection of Minimal Residual Disease in Blood Malignancy]

Real-time quantitative PCR (RQ-PCR) is a recently developed technique for nuclear acid quantification, in which conventional PCR and probe hybridization are integrated together. A specific fluorescent-labeled probe, binded with DNA/cDNA target, is supplemented into the reaction system of PCR. During the extension phase of PCR amplification, the specific fluorescent-labeled probe is cleaved by the 5'-->3' exonuclease activity of Taq polymerase, producing a fluorescent signal that can be detected. The amount of fluorescence released in the reaction is proportional to the target sequence number. Thus, an exactly and absolutely quantitative assessment of initial target DNA/cDNA copy number can be made in a closed tube assay system. This technique eliminates the need for post-PCR sample processing which is necessary in conventional PCR and decreases the probability of contamination which is inevitable in conventional PCR. Moreover, it reduces the potiental for false positive or negative results by utilizing the hybridization of a probe to sequences internal to amplification primers. RQ-PCR is proved to be a sensitive, specific, reliable and convenient tool for the quantification of minimal residual diseases (MRD) of hematological malignancies by recent studies. This article reviews the principle of RQ-PCR and its application in quantitative detection of MRD of hematological malignancies such as acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and malignant lymphoma.

Journal Article↗
PubMed2006

Unraveling transcription regulatory networks by protein-DNA and protein-protein interaction mapping.

Metazoan genomes contain thousands of protein-coding and noncoding RNA genes, most of which are differentially expressed, i.e., at different locations or at different times during development, function, or pathology of the organism. Differential gene expression is achieved in part by the action of regulatory transcription factors (TFs) that bind to cis-regulatory elements that are often located in or near their target genes. Each TF likely regulates many targets in the context of intricate transcription regulatory networks. Up to 10% of a genome may encode TFs, but only a handful of these have been studied in detail. Here, I will discuss the different steps involved in the mapping and analysis of transcription regulatory networks, including the identification of network nodes (TFs and their target sequences) and edges (TF-TF dimers and TF-DNA target interactions), integration with other data types, and network properties and emerging principles that provide insights into differential gene expression.

Animals↗
PubMed1994

Chimerization of antitumor antibodies via homologous recombination conversion vectors.

Homologous recombination vectors were designed to convert murine hybridoma cell lines expressing IgG3, IgG1, or IgG2a heavy chains into chimeric human IgG1 producers. These conversion vectors included homology both upstream and downstream of the target sequences and consistently resulted in a higher frequency of successful gene targeting than an insertion vector bearing a single region of homology. A human kappa light chain conversion vector was also constructed and used to complete chimerization of the anticarcinoma hybridoma cell line BR96. The resulting cell line expressed antigen-specific chimeric antibody at comparable levels to those found in the murine parental cell line. Southern blots confirm that recombination occurred within the upstream and downstream regions of homology for both vectors, resulting in the loss of murine constant region sequences.

Animals↗
PubMed2026

Cross-target and cell-preferential CRISPR-Cas9 inhibition with carbohydrate-tagged oligonucleotides.

Precise control of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) activity is important for limiting off-target effects and chromosomal rearrangements. Existing inhibitors, including anti-CRISPR proteins and spacer-targeting oligonucleotides, can be constrained by immunogenicity, target-sequence dependence, or delivery challenges. Here, we developed single-stranded DNA oligonucleotides that target conserved regions of the Streptococcus pyogenes Cas9 single-guide RNA (sgRNA) scaffold rather than the variable spacer. Screening identified single-stranded DNA 7 (ssDNA7), which targets stem-loop-1 and the adjacent linker and inhibits Cas9 activity across multiple tested spacer sequences and genomic loci without redesigning the inhibitor. Carbohydrate conjugation improved oligonucleotide stability and preferentially enhanced inhibitory activity in selected liver-derived or cancer cell models. Amplicon sequencing confirmed inhibition of endogenous editing in multiple cell models and normal liver-derived organoids, and supported cell-preferential inhibition in matched cell comparisons. These findings establish sgRNA-scaffold targeting as a strategy for cross-target inhibition of Cas9 and show that carbohydrate conjugation can tune its cellular activity.

CRISPR-Cas9↗
PubMed2005

RNA guanine quadruplex invasion by complementary and homologous PNA probes.

Guanine quadruplexes are gaining increasing attention due to their suspected roles in regulating gene expression at the transcriptional and translational levels. This paper describes the ability of short peptide nucleic acid (PNA) probes to disrupt a stable RNA quadruplex and hybridize to their target sequence. In one case, the PNA probe is complementary to the target, resulting in formation of a Watson-Crick base-paired duplex. In the second case, the PNA probe is homologous to the target and forms a hybrid quadruplex structure. The hybrid duplex is formed in a 1:1 stoichiometry, as expected based on the constraints imposed by Watson-Crick pairing. However, the hybrid quadruplex is formed in a PNA2:RNA stoichiometry, due to the ability of the short PNA to hybridize with both halves of the original RNA quadruplex.

Base Sequence↗
PubMed1994

Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.

Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibroblasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-ras proto-oncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 microM anti-BPDE showed a faster removal of the adducts from the H-ras gene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.

Animals↗
PubMed2007

Role of amniotic membrane graft for ocular chemical and thermal injuries.

PURPOSE: To evaluate the results of amniotic membrane transplantation (AMT) for ocular surface reconstruction in chemical and thermal injuries. METHODS: Retrospective review of case records of patients who had undergone AMT for chemical injuries (January 1998 to May 2001). RESULTS: Seventy two eyes of 69 patients were studied of which 24 were acute cases (median-2 days, range, 1-20 days) and 48 were chronic cases (median-12.4 months, range, 1.02-95.8 months). Mean age was 22.4 years (SD +/- 13.34 years) and average follow up duration was 7.8 months (SD +/- 7.1). Main clinical findings were symblephara (52.8%), corneal vascularization (51.3%), conjunctivalization (45.8%), Limbal ischemia (45.8%), Limbal stem cell deficiency (55.5%) and epithelial defect (48.6%). 18 cases were due to acid injuries (5 acute, 13 chronic), 52 were due to alkali (18 acute and 34 chronic) and 2 cases were due to thermal burns (1 each acute and chronic). Overall success rate was 87.5% in acute cases and 72.9% in chronic cases. Indication-wise success rates were 94.3% for epithelial defect healing, 88.2% for symptomatic relief, 59.7% for ocular surface reconstruction, and 55% for improving limbal stem cell function. Success was not achieved in any outcome measure in 1/24 (4.2%) in acute group and 6/48 (12.5%) in chronic group. CONCLUSION: AMT helps in ocular surface reconstruction, promotes rapid epithelial healing and partially restores limbal stem cell function. It can be considered as an effective modality for the ocular surface restoration in chemical and thermal injuries in selected cases. Success rates in acute and chronic cases are comparable.

Acute Disease↗