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At least 253 records · Page 14Linked to original sources
PubMed2000

Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells.

Adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on human chromosome 19q13.3-qter. Similarly, there is accumulating evidence that this site-specific integration occurs by transfection of AAV-based plasmid vectors. In order to further define the process of plasmid integration events, we constructed some AAV plasmids, introduced them into HeLa cells by lipofection, and isolated chromosomal integrants. One of such plasmids, pTH-5, contained the rep and neomycin-resistant (neo(r)) genes flanked by the 5'- and 3'-inverted terminal repeats of AAV and the hygromycin-resistant (hyg(r)) gene located in the plasmid backbone. Southern blot analysis revealed that among 36 G418-resistant (G418(r)) clones isolated, 22 (61%) showed site-specific integration into AAVS1. Further structural and functional analyses on the expression of the hyg(r) gene in the site-specific clones and the LacZ gene in clones generated with plasmid pTH-2 indicated that, together with the AAV sequence, the plasmid backbone was integrated into the AAVS1 site and thus the neo(r) and hyg(r) genes remained linked at high frequencies in the targeted integrants compared with random integrants. Sequence analysis of integration junctions between pTH-5 and AAVS1 revealed that the junctions occurred in the p5 promoter region of the plasmid while mainly in the partial cDNA coding region of the AAVS1 site. We also found that plasmid pTH-1 linearized in the backbone before lipofection gave a significantly lower frequency of site-specific integration (26%) than the circular form (60%). This finding may support the involvement of the double-stranded, circular form of infected AAV in the integration process. Our results may help to understand the process and mechanism of site-specific integration of lipofected AAV plasmid vectors.

Cation Exchange Resins↗
PubMed1991

Introduction of single-stranded ADH genes into Drosophila results in tissue-specific expression.

We injected single-stranded circular DNA containing a Drosophila Adh gene into ADH-negative embryos of Drosophila melanogaster and performed ADH histochemical staining on third instar larvae of the injected generation. Introduction of either the coding or non-coding strand resulted in correct tissue-specific expression of the Adh gene in larvae. Southern blotting revealed that the bulk of the injected DNA became double-stranded shortly after injection and was not integrated into the genome.

Alcohol Dehydrogenase↗
PubMed2026

Field-isolate recombinant tick-borne encephalitis viruses define reporter-stability guidelines for antiviral testing in flaviviruses.

As arthropod-borne viruses continue to threaten populations globally, there is a pressing need for experimental systems that enable rapid antiviral discovery. Reverse-genetics platforms producing recombinant reporter orthoflaviviruses have been developed to address this gap. Here, we present two new recombinant tick-borne encephalitis viruses (TBEVrec) generated on a European-subtype Haselmühl Tiho1 isolate backbone. A reporter gene, either eGFP or Nluc, was inserted in the capsid-coding region of the genome downstream of the capsid RNA regulatory signal and separated from the complete viral polyprotein by a 2A self-cleaving peptide. TBEVrec was better rescued using the circular polymerase extension reaction (CPER) than with the infectious subgenomic amplicon (ISA) method. TBEVrec replicated efficiently in relevant human cell lines, with comparable replication to wild-type TBEV in a neuronal cell line and moderately reduced titers and RNA levels in immune-derived cell lines. Using either eGFP or Nluc, we illustrate how TBEVrec enabled high-content RNAi screening, highlighting Nucleolin and PRKD1 as potential TBEV host factors, and drug testing on a benchtop plate reader. Nanopore sequencing of the eGFP insert revealed that the reporter is excised without affecting flanking regions. Comparative analysis of eGFP and Nluc further shows that this instability is time- and cell type-dependent, and that Nluc is comparatively more stable. From these observations, we outline safeguards and design principles that are broadly applicable both to the rescue of existing constructs and to the design of future recombinant reporter virus platforms.

CPER↗
PubMed1991

Splice site requirement for the efficient accumulation of polyoma virus late mRNAs.

Polyoma virus late nuclear primary transcripts are giant and heterogeneous, containing tandem repeats of the late strand of the circular viral genome. Late pre-mRNA processing involves the splicing of noncoding 'leader' exons to each other (removing genome-length introns), with the joining of the last leader to a coding 'body' exon. We have constructed a number of mutants blocked only in leader-leader splicing, or blocked in both leader-leader and leader-body splicing. We examined the accumulation of both nuclear and cytoplasmic late-strand RNAs in NIH3T3 cells. Consistent with our previous results, mutants lacking the 3' splice site of the late leader (leader-leader splicing blocked) showed a 10-20 fold defect in late RNA accumulation. Mutants which lacked the leader 5' splice site (leader-body splicing blocked) had a more profound defect, exhibiting virtually no late-strand cytoplasmic or nuclear RNA. This result was unexpected as a substantial proportion of wild type late cytoplasmic messages are unspliced. A mutant with no intron, but having functional 3' and 5' splice sites bordering the leader exon, is capable of producing large amounts of unspliced late mRNA. This demonstrates that an excisable intron is not a requirement for late mRNA accumulation. The accumulation of polyoma late mRNAs requires the presence of leader exons bordered by functional 3' and 5' splice sites, whether or not these sites are used during pre-mRNA processing.

Base Sequence↗
PubMed1978

[Characterization of plasmid DNA in a lecithinase-positive and in a lecithinase-negative strain of Clostridium perfringens (author's transl)].

A non pathogenic variant of Clostridium perfringens and the wild type strain were characterized. The strains agreed in most of the biochemical properties, in susceptibility against antibiotics and in bacteriocin production. Contrary to the wild type the variant did not produce lecithinase and gelatinase. In deoxyribonucleic acid (DNA) of both strains centrifuged in cesiumchlorid-ethidiumbromide equilibrium there was found a satellite peak containing three distinct, covalently closed circular (CCC) DNA elements. The sum of the average molecular weight or contour length of the two small circular molecules was equal to the average molecular weight or contour length of the third. The presence of the plasmids in the variant indicated that the synthesis of lecithinase might not be coded by a plasmid.

Bacteriocins↗
PubMed1996

Infectivity and complete nucleotide sequence of the genome of a genetically distinct strain of maize streak virus from Reunion Island.

A complete infectious genome of an isolate of maize streak subgroup 1 geminivirus from Reunion Island (MSV-R) was cloned and sequenced. Using an Agrobacterium tumefaciens Ti plasmid delivery system, the cloned 2.7 kb circular DNA was shown to be infectious in maize. The agroinfected virus could be transmitted by Cicadulina mbila, the most common vector species of MSV in Reunion. Analysis of open reading frames (ORFs) revealed seven potential coding regions including the 4 ORFs conserved in all geminiviruses infecting monocotyledonous plants, the 2 on the viral "+" strand (MP, CP), and the 2 on the complementary "-" strand (RepA, RepB). The nucleotide sequence of MSV-R was compared to previously determined sequence of three African clones from Nigeria (MSV-N), Kenya (MSV-K), and South Africa (MSV-S). More similarity was found between the African clones (97.0-97.3%) than between these and MSV-R (94.4-95.3%). Nucleotide substitutions were frequent in the large intergenic region, particularly in and around the most likely TATA box for the complementary sense genes, and in the 5' end of ORF V1. The comparison of the predicted peptide sequences of the proteins encoded by ORFs MP, RepA and RepB confirmed the higher similarity between the African clones (97.8-99.3%) than between these and MSV-R (95.1-97.1%). However the amino acid sequences of the protein encoded by ORF CP (capsid protein) were very conserved among all the 4 clones, suggesting a high selection pressure on this ORF.

Base Sequence↗
PubMed1992

Protein-coding introns from the 23S rRNA-encoding gene form stable circles in the hyperthermophilic archaeon Pyrobaculum organotrophum.

Two archaeal introns have been discovered in the single-copy 23S rRNA-encoding gene of the hyperthermophile, Pyrobaculum organotrophum. After excision from rRNA transcripts, both introns circularize and are stably retained in the cell. Putative proteins encoded by the introns and covering most of the intron sequence share a decapeptide motif with proteins encoded by another archaeal intron and by group I introns.

Amino Acid Sequence↗
PubMed1990

Hepatitis A, B, C, D and E viruses: structure of their genomes and general properties.

Hepatitis A virus is an enteric picornavirus. Its genome is a single stranded RNA molecule of positive-strand polarity of 7478 bases. This sequence codes for a polyprotein which is processed to give rise to viral proteins VP-1, VP-2, VP-3 and others. Hepatitis B virus, a major worldwide infectious and cancer promoting agent contains a DNA genome of 3226 base pairs that replicates by a reverse transcriptase via an RNA intermediate. Extensive sequencing and expression experiments have revealed four major genes named surface, core, polymerase and X which are coded in more than one reading frame. Furthermore, within a frame, proteins are expressed from multiple initiation codons resulting in several related products. The viral genome of hepatitis C virus (nonA-nonB), an elusive major infectious agent, has recently been cloned. This genome is a single positive-stranded RNA of at least 10,000 bases which codes for several antigens, some of them associated specifically with nonA-nonB hepatitis infections. The hepatitis D (delta) viral agent, an infectious agent requiring a hepadnarious for propagation, contains a covalently closed circular single-stranded RNA genome of 1167 nucleotides. This genome encodes the protein p24 and p27 that bind specifically to antisera from patients with chronic hepatitis D infections.

Genes, Viral↗
PubMed1979

Characterization of a histone-like protein extracted from yeast mitochondria.

Analysis of proteins isolated by affinity chromatography on DNA-cellulose from highly purified yeast mitochondria shows that these organelles do not contain histones but have in abundance a DNA-binding protein of 20,000 daltons. The purification yield of this protein, called HM, indicates that mitochondria have at least an equal mass of HM relative to DNA. The amino acid composition and its electrophoretic characterization reveal that HM, rich in lysine, is slightly basic and heat stable. HM appears to be coded by the yeast nucleus, as shown by its presence in several "petite" mutants. We have shown that HM, like histones or histone-like proteins, is able to introduce superhelical turns into circular relaxed DNA in the presence of a nicking-closing activity.

Amino Acids↗
PubMed2026

Complete mitochondrial genomic sequence of Lenzites betulinus (Polyporales, Basidiomycota).

Lenzites betulinus is a medicinal fungus in the Polyporaceae. Here, we report the first complete mitochondrial sequencing of this species. The circular genome is 61,288 bp in length, with a GC content of 26.32% and base composition of A (36.98%), T (36.70%), G (12.96%), and C (13.36%). It contains 14 core protein-coding genes (PCGs), 26 tRNA genes, two rRNA genes, and nine intronic ORFs within the cox1 gene. A maximum-likelihood phylogenetic tree based on 14 PCGs from 17 mitochondrial genomes confirmed that L. betulinus clusters within the Polyporaceae, closely related to Trametes and Fomitopsis. This served as a significant reference for ongoing research into other species within the Polyporales order.

Mitochondrial genome↗
PubMed2000

Complete nucleotide sequence and characterization of pSNA1 from pimaricin-producing Streptomyces natalensis that replicates by a rolling circle mechanism.

A cryptic plasmid, pSNA1, has been identified in the pimaricin-producing Streptomyces natalensis strain ATCC 27448. pSNA1 has been mapped with restriction endonucleases and its complete nucleotide sequence was determined. The circular DNA molecule is 9367 bp in length and has a 71.3% G+C content. Its estimated copy number is 30. Analysis of the sequence and codon preferences indicated that pSNA1 contains seven open reading frames [encoding peptides larger than 90 amino acid (aa) residues], ORF 1 to ORF 7, located on both strands of pSNA1. ORF 3 codes for a protein (476 aa) that shows high sequence similarity to replication-associated proteins in Streptomyces plasmids known to replicate via the rolling circle mechanism. Accumulation of single-strand intermediates further indicates that pSNA1 replicates via the rolling circle replication model. ORF 1 encodes a polypeptide of 246 aa that shares homology with KorA proteins encoded by other streptomycete plasmids. ORF 4 (SpdA) codes for a protein (161 aa) possibly involved in intramycelial plasmid transfer. Protein encoded by ORF 2 (309 aa) shares homology with a Streptomyces protein (SpdB2) also involved in plasmid spreading.

Amino Acid Sequence↗
PubMed1988

Thymocyte circular DNA excised from T cell receptor alpha-delta gene complex.

We have characterized thymocyte circular DNA excised from the T cell receptor alpha-delta gene complex. Some delta gene clones contained unusual recombinant structures derived from V-(D)-J joining: (i) a reciprocal joint of direct V to J delta joining, skipping the D delta segment; (ii) a V-D delta coding joint lacking an adjacent D delta-J delta coding joint; (iii) a V- D structure containing two D delta segments. Many of the alpha gen clones contained both coding and reciprocal joints of V alpha-to-J alpha joining on the same structure. Most of these coding joints were out of phase; however, in one clone there was an in-phase V-J alpha structure. Interestingly, some alpha gene clones contained the same V gene sequence as rearranged in the delta gene clone, indicating that the same V gene family, at least in part, could be utilized for both the alpha and delta gene systems.

Amino Acid Sequence↗
PubMed1995

Morphological changes during ontogeny of the canine proximal colon.

The development of the canine proximal colon from the completion of organogenesis through 43 days after birth was studied using light microscopy, immunofluorescence and electron microscopy. During this period the tunica muscularis increased in thickness from 42+/-6 microm in animals midway through the gestation period to 317+/-29 microm in animals 25-30 days old. This increase in thickness resulted from an increase in the number and size of smooth muscle cells in the circular and longitudinal muscle layers. The cross-sectional thickness of the circular muscle layer increased from 10+/-2 smooth muscle cells midway through the gestation period to 92+/-7 cells in animals 25-30 days old. The longitudinal layer increased in thickness from 1.5+/-1 cells in animals midway through the gestation period to 44+/-2 cells in animals 25-30 days old. Smooth muscle cells from both layers also increased in diameter and length. Ultrastructural and immunohistochemical studies suggested that many of the smooth muscle cells were undergoing development throughout the fetal period. Midway through the gestation period, the circular layer was positive for desmin-like immunoreactivity (D-LI), while both the circular and longitudinal layers were positive for vimentin-like immunoreactivity (V-LI). By birth, V-LI was suppressed in the circular and longitudinal layers, and both layers expressed D-LI. The enteric nervous system was already established midway through the gestation period, and submucosal and myenteric ganglia could be identified, although the chemical coding and mature morphology of neurons were incomplete. NADPH-diaphorase-positive neurons, indicating the expression of nitric oxide synthase, developed by the time of birth. Interstitial cells of Cajal (IC) could not clearly be identified midway through gestation, however, potential precursors to ICs were observed. Several classes of ICs were identifiable at birth.

Animals↗
PubMed1979

Stable yeast transformation with chimeric plasmids using a 2 micron-circular DNA-less strain as a recipient.

By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 micron yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 micron DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 micron-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura+ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5' monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type. These features should offer new possibilities for cloning with yeast.

Chimera↗
PubMed1995

The stability of tropomyosin, a two-stranded coiled-coil protein, is primarily a function of the hydrophobicity of residues at the helix-helix interface.

The sequences of coiled coils are characterized by a repeating heptad of amino acids, abcdefg, in which the a and d residues are generally hydrophobic and form the interface between the two alpha-helices. In this study, rat and chicken alpha-tropomyosins (alpha-TMs) have been used as models to determine whether the effects of mutations on the stability of two-stranded coiled coils can be predicted by a simple algorithm. The thermal stabilities of three wild-type muscle alpha-TMs and nine chimeras, in which the second and/or sixth or ninth coding exons of one alpha-TM cDNA were replaced with exons from other alpha-TM cDNAs, with a sequence encoding the GCN4 leucine zipper or a random coil sequence, have been obtained using circular dichroism spectroscopy. Tropomyosin is almost completely helical along its entire length, but there is no correlation of the thermal stability of the alpha-TMs with the helical propensity of their component amino acids. The stability can be predicted (P = 0.90), however, by assigning a weight to every amino acid residue in each sequence, depending on its frequency of occurrence at the abcdef or g position in a data base of coiled-coil fibrous proteins, and summing all the weights. The correlation improves if only the residues at the a and d interface are counted (P = 0.94). The major factor modulating the thermal stability appears to be the hydrophobicity of the residues at the coiled-coil interface, since there is a high correlation (P = 0.91) of the TM values with the sum of the hydrophobic moments of the residues found at the a and d positions.

Algorithms↗
PubMed2000

Species-specific TT viruses and cross-species infection in nonhuman primates.

Viruses resembling human TT virus (TTV) were searched for in sera from nonhuman primates by PCR with primers deduced from well-conserved areas in the untranslated region. TTV DNA was detected in 102 (98%) of 104 chimpanzees, 9 (90%) of 10 Japanese macaques, 4 (100%) of 4 red-bellied tamarins, 5 (83%) of 6 cotton-top tamarins, and 5 (100%) of 5 douroucoulis tested. Analysis of the amplification products of 90 to 106 nucleotides revealed TTV DNA sequences specific for each species, with a decreasing similarity to human TTV in the order of chimpanzee, Japanese macaque, and tamarin/douroucouli TTVs. Full-length viral sequences were amplified by PCR with inverted nested primers deduced from the untranslated region of TTV DNA from each species. All animal TTVs were found to be circular with a genomic length at 3.5 to 3.8 kb, which was comparable to or slightly shorter than human TTV. Sequences closely similar to human TTV were determined by PCR with primers deduced from a coding region (N22 region) and were detected in 49 (47%) of the 104 chimpanzees; they were not found in any animals of the other species. Sequence analysis of the N22 region (222 to 225 nucleotides) of chimpanzee TTV DNAs disclosed four genetic groups that differed by 36.1 to 50.2% from one another; they were 35.0 to 52.8% divergent from any of the 16 genotypes of human TTV. Of the 104 chimpanzees, only 1 was viremic with human TTV of genotype 1a. It was among the 53 chimpanzees which had been used in transmission experiments with human hepatitis viruses. Antibody to TTV of genotype 1a was detected significantly more frequently in the chimpanzees that had been used in transmission experiments than in those that had not (8 of 28 [29%] and 3 of 35 [9%], respectively; P = 0.038). These results indicate that species-specific TTVs are prevalent in nonhuman primates and that human TTV can cross-infect chimpanzees.

Animals↗
PubMed1991

Stable submolecular folding units in a non-compact form of cytochrome c.

Studies of structure, dynamics, and stability of cytochrome c (cyt c) at low pH in a non-compact pre-molten globule state indicate that the protein contains submolecular folding units that are independently stable. In high salt, acid cyt c (pD 2.2; where D is deuterium) is nearly as compact as the native form. Nuclear magnetic resonance (n.m.r.) line broadening typical of the molten globule form is seen, indicating loosened packing and increased mobility not only for side-chains but also for the main chain. As NaCl concentration is decreased below 0.05 M, cyt c expands due to the deshielding of electrostatic repulsions, attaining a linear extent perhaps double that of the native protein (viscosity, fluorescence). In the extended form, tertiary structural hydrogen bonds are largely broken (hydrogen exchange rate), some normally buried parts of the protein are exposed to water (fluorescence), and many of the native side-chain contacts must be lost. Nevertheless, almost all of the helical content is retained (circular dichroism). The helices involve the same amino acid residues that are helical in the native state (hydrogen exchange labeling monitored by 2-dimensional n.m.r.). The equilibrium constant for helix formation at 20 degrees C (0.02 M-NaCl, pD 2.2) is about 10 (hydrogen exchange rate), even though the individual helical segments when isolated have little or no structure. Additional experiments were done to check assumptions and calibrate parameters that underlie the hydrogen exchange analysis of protein folding. These results indicate that the native-like helical segments in the expanded non-globular form of cyt c exist as part of somewhat larger submolecular folding units that possess significant equilibrium stability. Results from equilibrium and kinetic studies of protein folding support the generality of this conclusion. This view is contrary to the two-state paradigm for equilibrium folding and inconsistent with the idea that side-chain packing constraints determine folding motifs. The result suggests an extension of the thermodynamic hypothesis for protein structure to kinetic folding processes, so that the amino acid code for equilibrium and kinetic folding may be the same, and also seems pertinent to the biological evolution of contemporary protein structures.

Chromatography, Gel↗