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At least 361 records · Page 20Linked to original sources
PubMed2001

Calreticulin, a calcium-binding molecular chaperone, is required for stress response and fertility in Caenorhabditis elegans.

Calreticulin (CRT), a Ca(2+)-binding protein known to have many cellular functions, including regulation of Ca(2+) homoeostasis and chaperone activity, is essential for heart and brain development during embryogenesis in mice. Here, we report the functional characterization of Caenorhabditis elegans calreticulin (crt-1). A crt-1 null mutant does not result in embryonic lethality but shows temperature-dependent reproduction defects. In C. elegans CRT-1 is expressed in the intestine, pharynx, body-wall muscles, head neurons, coelomocytes, and in sperm. crt-1 males exhibit reduced mating efficiency and defects late in sperm development in addition to defects in oocyte development and/or somatic gonad function in hermaphrodites. Furthermore, crt-1 and itr-1 (inositol triphosphate receptor) together are required for normal behavioral rhythms. crt-1 transcript level is elevated under stress conditions, suggesting that CRT-1 may be important for stress-induced chaperoning function in C. elegans.

Animals↗
PubMed1983

Immunocytochemical characterization of human blood cells.

A simple immunocytochemical method using calf intestinal alkaline phosphatase as the enzymatic indicator to demonstrate surface antigens on human blood cells has been developed. The blood cells were labeled with cell specific monoclonal antibodies followed by linkage with an antiimmunoglobulin alkaline phosphatase conjugate. Cytochemical demonstration of alkaline phosphatase activity on the blood cells reflects the presence of surface antigens on these cells. The effects on the cytochemical reaction of fixation, substrates, couplers, activators and inhibitors, and storage of cytologic materials have been examined systematically. The best staining conditions are to incubate labeled smears in a 0.04 M barbital buffer at pH 7.6 containing 30 mg% naphthol AS-TR phosphate, 40 mg% fast red ITR, and 1 mM levamisole. This method is both sensitive and specific and appears most practical for objective identification of the human blood cells.

Alkaline Phosphatase↗
PubMed2002

Genetic dissection of the initiation of the infection process and nodule tissue development in the Rhizobium-pea (Pisum sativum L.) symbiosis.

Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.

Meristem↗
PubMed2026

ALPINE: a scalable pipeline for comprehensive classification of gene-editing outcomes from long-read amplicon sequencing.

SUMMARY: CRISPR genome editing has enabled precise genetic modification for gene and cell therapies, but edits often produce heterogeneous on-target outcomes, including homology-directed repair (HDR) knock-ins, DNA repair template integrations, and structural variants. Existing tools are frequently limited to short reads or lack viral vector-specific integration categories needed for therapeutic development. Here, we present ALPINE (Amplicon Long-read Pipeline for INtegration Evaluation), a scalable and reproducible pipeline for classifying and quantifying gene-editing outcomes from long-read amplicon sequencing supporting both PacBio HiFi and Oxford Nanopore platforms. ALPINE classifies reads into 10+ categories, including DNA repair vector integration subtypes, and performs variant calling near the gene-edited site with batch, multi-sample reporting. Uniquely, ALPINE can distinguish between cells treated with multiple DNA repair vectors and identify distinct molecular features, such as inverted terminal repeats (ITRs), enabling comprehensive characterization of complex gene editing outcomes. Dual-target benchmarking on simulated datasets demonstrated high accuracy for transgene integration events. Independent validation on public crosslinked-HDR dataset confirmed ALPINE's integration detection capabilities, and application to edited T cell samples demonstrated comprehensive gene-editing outcome profiling. AVAILABILITY: ALPINE is available under MIT license at https://github.com/Maggi-Chen/ALPINE and https://doi.org/10.5281/zenodo.20272510. All analysis scripts and visualization code used in this manuscript are available at https://github.com/Maggi-Chen/ALPINE-manuscript-analysis. Simulated datasets are deposited at Zenodo (https://doi.org/10.5281/zenodo.20260865). Public dataset PRJNA913199 is available through NCBI SRA.

Gene Editing↗
PubMed1990

The transposable element Uhu from Hawaiian Drosophila--member of the widely dispersed class of Tc1-like transposons.

We report the complete nucleotide sequence of the transposable element Uhu from the vicinity of the alcohol dehydrogenase (Adh) gene of Drosophila heteroneura (an endemic Hawaiian Drosophila). The complete element is about 1650 base-pairs (bp) long, has 46-50 base-pair inverse imperfect repeats at it's ends, and contains a large open reading frame potentially encoding a 192 amino acid protein. We demonstrate that Uhu belongs to a class of transposable elements which includes Tc1 from Caenorhabditis elegans, Barney from Caenorhabditis briggsae, and HB1 from Drosophila melanogaster. All of these elements share significant sequence similarity, are approximately 1600 base pairs long, have short inverse terminal repeats (ITRs), contain open reading frames (ORFs) with significant sequence identity, and appear to insert specifically at TA sequences generating target site duplications.

Alcohol Dehydrogenase↗
PubMed1999

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2.

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.

Base Sequence↗
PubMed1991

Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat.

The nucleotide sequence of 42090 bp of vaccinia virus strain WR is presented. The sequence includes the SalI L, F, G and I fragments and starts near the centre of the HindIII A fragment and extends rightwards towards the genomic terminus, finishing approximately 0.5 kb internal of the inverted terminal repeat (ITR). Translation of this region has identified 65 open reading frames (ORFs) of greater than 65 amino acids in length. Fifty-one of these which do not extensively overlap other larger ORFs have been subjected to further analysis; the other 14 are termed minor ORFs. In the rightmost 28.7 kb, the genes are, with one exception, transcribed towards the genomic terminus, similar to the arrangement of genes at the left end of the virus genome. Internal of this region the genes are expressed off either DNA strand but still predominately rightwards. ORFs are tightly packed with few intergenic non-coding regions of greater than 250 bp. Protein sequence comparisons have established a remarkably high number of homologies with entries in existing protein databases. Of these, DNA ligase, thymidylate kinase, two serine-threonine protein kinases, two serine proteinase inhibitors (serpins), two interleukin-1 receptor homologous and a discontinuous ORF related to tumour necrosis factor receptor have been reported. Other homologies include lectins, profilin, 3 beta-hydroxy steroid dehydrogenase, superoxide dismutase, guanylate kinase, ankyrin and complement factor H. In addition, there are a number of polypeptides with predicted properties of membrane-associated, secretory or glyco-proteins. Twelve gene families are described here and elsewhere. There is considerable similarity between genes from the right and left end of the virus genome that may have arisen by terminal transposition events. Several differences from the corresponding region of vaccinia virus strain Copenhagen sequence are noted. Near the right terminus the sequences diverge completely, and internal of this there are multiple examples of deletion of short sequences (eight to 10 nucleotides) that lie within penta- or hexanucleotide direct repeats.

Amino Acid Sequence↗
PubMed2001

A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors.

The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells. Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest. We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV). AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method. We show here that co-infection of mammalian producer cells with three viruses - a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene - produces infectious rAAV particles. This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production.

Baculoviridae↗
PubMed1999

Localization of mariner DNA transposons in the human genome by PRINS.

Homologous recombination occurring among misaligned repeated sequences is a significant source of the molecular rearrangements resulting in human genetic disease. Studies of the Charcot-Marie-Tooth disease locus on chromosome 17 have implicated the involvement of an ancient DNA transposon of the mariner family (Hsmar2) in the initiation of double-strand break events leading to homologous recombination. In this study, the genomic locations of 109 Hsmar2 elements were determined by primed in situ labeling (PRINS) using primers designed to match the right and left inverted terminal repeats (ITRs) of the transposon. Although the resolution of the PRINS technique is approximately 400 chromosomal Giemsa bands, the data presented here provide the first large-scale mapping study of these elements, which may be involved in initiation of homologous recombination events in the human genome.

Chromosome Mapping↗
PubMed1993

Differential regulation of two myo-inositol transporter genes of Saccharomyces cerevisiae.

We previously isolated two distinct Saccharomyces cerevisiae myo-inositol transporter genes, ITR1 and ITR2 (Nikawa et al., 1991). Here, we studied the regulation of their expression by measuring steady-state mRNA levels and beta-galactosidase activities of lacZ fusion genes under different conditions. The results show that the expression of the two ITR genes is differently regulated: ITR1 was repressed by inositol and choline whereas ITR2 was constitutive. Deletion analysis of the ITR1 upstream region and comparison with the upstream regions of other genes involved in phospholipid synthesis indicate that the octamer sequence 5'-TTCACATG-3' is important for the expression and inositol/choline regulation of the ITR1 gene.

Amino Acid Sequence↗
PubMed2006

Expression profiles of bovine adeno-associated virus and avian adeno-associated virus display significant similarity to that of adeno-associated virus type 5.

We present the first detailed expression profiles of nonprimate-derived adeno-associated viruses, namely, bovine adeno-associated virus (B-AAV) and avian adeno-associated virus (A-AAV), which were obtained after the infection of cell lines derived from their natural hosts. In general, the profiles of B-AAV and A-AAV were quite similar to that of AAV5; however, both exhibited features found for AAV2 as well. Like adeno-associated virus type 5 (AAV5), B-AAV and A-AAV utilized an internal polyadenylation site [(pA)p]; however, it was used to greater relative levels by B-AAV than by A-AAV. Similar to AAV5, >99% of B-AAV RNAs generated from upstream promoters were polyadenylated at (pA)p and hence not spliced. In contrast, ca. 50% of the A-AAV RNAs generated from upstream promoters read through (pA)p, as seen for AAV2. However, A-AAV generated lower levels of spliced P5 and P19 products than does AAV2, suggesting that A-AAV generates lower relative levels of Rep 68 and Rep 40. An additional difference in the expression profile of these viruses was that B-AAV generated a greater level of ITR-initiated RNAs than did A-AAV or AAV5. In addition, we demonstrate that, like AAV2, transactivation of transcription of the capsid-gene promoter of B-AAV required both adenovirus and targeting of its Rep protein to the transcription template; however, expression of the capsid-gene promoter of A-AAV was, like AAV5, largely independent of both adenovirus and its Rep proteins.

Animals↗
PubMed1998

Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue.

Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.

Animals↗
PubMed2001

Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer.

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.

Animals↗
PubMed2005

Cation effect on thermal transition of iota-carrageenan: a photon transmission study.

Coil-to-double helix (c-h) and double helix-to-dimer (h-d) phase transitions of iota-carrageenan in various cation (CaCl2) solutions upon heating and cooling were studied using the photon transmission technique. Photon transmission intensity, Itr, was monitored against temperature to determine the (c-h) and (h-d) transition temperatures (Tch and Thd) and activation energies (DeltaEch and DeltaEhd). An extra dimer-to-dimer (d-d) transition was observed during cooling at low temperature region. However, upon heating dimers directly decompose into double helices by making dimer-to-double helix (d-h) transition. Further heating results in double helix-to-coil (h-c) transition at high temperature region. Tdh and Thc temperatures and Delta Edh and DeltaEhc activation energies were determined. It was observed that Thc and Tch temperatures and DeltaEch and DeltaEhc activation energies were found to be strongly correlated to the CaCl2 content in the system.

Biocompatible Materials↗
PubMed2003

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway.

A systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2- and AAV-5-based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5alpha dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.

Androgens↗
PubMed1984

A new bronchofiberscope for the study of diseases of very peripheral airways.

A new device, the BF-1.8T, has been designed to go through the 2.6 mm channel of the conventional fiberoptic bronchoscope (Olympus BF type ITR). It measures 1.8 mm in outer diameter; it has a visual angle of 75 degrees, a range of observation of 3 to 30 mm, an effective length of 950 mm, and a total length of 1120 mm. The tip can extend from the wedge of the outside endoscope by up to 180 mm, providing safe manipulation by the examiner. The results of four cases were briefly presented out of nearly 100 so far examined in our hospital. Selective alveolobronchography (SAB) was simultaneously performed because SAB could be done easily and the results were complementary to the direct observation which was obtained with the BF-1.8T. These data were also correlated with the results of transbronchial lung biopsies.

Adult↗
PubMed2001

Specific nucleoprotein complexes within adenovirus capsids.

Adenoviral DNA was examined within capsids by dimethyl sulfate footprinting. Protein-DNA interactions were visualized through ligation-mediated PCR (LM-PCR). Signals for protein binding were found adjacent to both inverted terminal repeats (ITR). There were no indications of close protein binding at several other loci of the viral genome. Therefore, adenovirus type 5 seems to contain sequence- or locus-specific DNA binding proteins within the virion.

Adenoviridae↗
PubMed2006

Chimeric herpes simplex virus/adeno-associated virus amplicon vectors.

Chimeric or hybrid herpes simplex virus type 1/adeno-associated virus amplicon vectors combine the large transgene capacity of HSV-1 with the potential for site-specific genomic integration and stable transgene expression of AAV. These chimeric vectors have been demonstrated to support transgene expression for significantly longer periods than standard HSV-1 amplicons. Moreover, HSV/AAV hybrid vectors can mediate integration at the AAVS1 pre-integration site on human chromosome 19 at a relatively high rate, although random integration has also been observed. One major remaining hurdle of HSV/AAV hybrid vectors is the low packaging efficiency and titers when AAV rep sequences are included in the amplicon vector. In the conditions prevalent during the replication/packaging of HSV/AAV hybrid amplicons into HSV-1 virions, in particular the presence of HSV-1 replication factors and AAV Rep protein, at least three different viral origins of DNA replication are active: the HSV-1 ori, the AAV inverted terminal repeats (ITRs), and the p5 promoter/ori driving expression of the AAV rep gene. A detailed understanding of the properties of these origins of DNA replication and the molecular mechanisms of interactions between them, may allow designing novel hybrid vectors that allow the efficient and precise integration of large transgenes in the human genome.

Dependovirus↗