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At least 181 records · Page 10Linked to original sources
PubMed1993

Structure/function studies of HIV-1(1) reverse transcriptase: dimerization-defective mutant L289K.

Virion-derived HIV-1 reverse transcriptase (RT) has subunits of molecular mass 66 and 51 kDa (p66 and p51, respectively) in an approximately 1:1 ratio. Since enzyme activity appears to depend on dimerization of these subunits, identification of critical regions of primary sequence required for proper dimerization could lead to potential targets for antiviral therapy. A central region of primary sequence contains a leucine hepta-repeat motif from leucine 282 to leucine 310 that has been suggested to be involved in dimerization [Baillon, J. G., Nashed, N. T., Kumar, A., Wilson, S. H., & Jerina, D. M. (1991) New Biol. 3, 1015-1019]. A region including this hepta-repeat was recently shown to be involved in protein-protein interactions required for dimerization [Becerra, S. P., Kumar, A., Lewis, M. S., Widen, S. G., Abbotts, J., Karawya, E. M., Hughes, S. H., Shiloach, J., & Wilson, S. H. (1991) Biochemistry 30, 11708-11719]. To investigate the role of this repeat motif in dimerization, we performed site-directed mutagenesis of these leucine residues from position 282 to position 310. Mutations were introduced into p66 and p51 RT coding sequences, and the individually purified RT subunit polypeptides were compared with wild-type polypeptides for dimerization. Physical characterization of the purified mutant peptides was conducted by circular dichroism analysis. Binding between p66 and p51 was studied by gel filtration, ultracentrifugation, and CD analysis. L289K-p66 was unable to dimerize with itself and wild-type or L289K-p51. The leucine repeat motif in the p66 subunit appears to be critical in formation of the heterodimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Morphogenesis of recombinant HIV-2 gag core particles.

The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.

Base Sequence↗
PubMed1981

Endodeoxyribonuclease activity associated with Rauscher murine leukemia virus.

Preparations of purified Rauscher murine leukemia virus were found to contain an endodeoxyribonuclease after disruption of the virus with nonionic detergents. The enzyme makes single-strand breaks in linear or covalently closed circular phage double-stranded DNA molecules. The enzyme was partially purified by ion-exchange chromatography on DEAE- and carboxymethyl-Sepharose columns followed by electrophoresis in DNA-containing polyacrylamide gels. The enzyme was separated from reverse transcriptase (p80pol), and the final endonuclease preparation contained no detectable reverse transcriptase activity. The DEAE-Sepharose column-purified endonuclease activity contained a polypeptide of about 40,000 Mr that we term p40. Peptide mapping experiments demonstrated that p40 shares methionine-labeled tryptic peptides with Pr200gag-pol and Pr135pol. Six major methionine-labeled tryptic peptides derived from p40 were found in Pr200gag-pol, but only five of these were detected in Pr135pol. The four core proteins (p30, p15, pp12, and p10) and p80pol plus p40 account for most, but not all, of the peptide sequences of Pr200gag-pol. The endonuclease-associated p40 is similar in size and precursor origin to the avian retrovirus-coded endonuclease (p32). In view of these similarities to the avian p32 endonuclease and its association with partially purified Rauscher murine leukemia virus-associated endonuclease preparations, we propose that p40 is the Rauscher murine leukemia virus-coded endonuclease.

Chromatography, Ion Exchange↗
PubMed2026

Cancer-associated fusion transcripts: mechanisms, functional roles, and clinical implications.

Fusion transcripts are hybrid RNA molecules generated through genomic rearrangements or RNA-level fusion mechanisms. They represent important molecular features of many cancers and can function as oncogenic drivers, diagnostic biomarkers, prognostic indicators, and therapeutic targets. Since the discovery of the BCR::ABL1 fusion in chronic myeloid leukemia, numerous cancer-associated fusion transcripts have been identified across hematologic malignancies and solid tumors. These fusion events encompass diverse biological mechanisms, including constitutively active kinases, aberrant transcription factors, epigenetic regulators, and non-coding fusion RNAs. This review summarizes current knowledge of the mechanisms underlying fusion transcript formation, including genomic rearrangement-dependent and rearrangement-independent processes, as well as fusion circular RNAs. The functional roles of fusion transcripts in cancer biology and their clinical relevance as diagnostic, prognostic, and predictive biomarkers are discussed. In addition, recent advances in fusion transcript detection and characterization are reviewed, including next-generation sequencing, long-read sequencing, single-cell approaches, artificial intelligence-assisted computational methods, and CRISPR/Cas9-mediated strategies for functional modeling and functional validation of fusion transcripts. Despite the rapid expansion of fusion transcript catalogs, the biological and clinical significance of most identified fusion events remains incompletely understood. Future progress will depend on integrating advanced sequencing technologies, artificial intelligence-assisted computational prioritization, and systematic functional validation to distinguish clinically actionable fusion transcripts from biologically neutral events. Such multidisciplinary approaches will be essential for translating fusion transcript research into precision oncology and improving cancer diagnosis, patient stratification, and targeted therapy.

Humans↗
PubMed1988

Description of the scatter component in photon-beam data.

Kerma scatter-to-primary ratios (SPR) were calculated, using a Monte Carlo code, for water irradiated by photon beams (200 keV monoenergetic, 60Co gamma, 4 and 15 MV x-rays). The SPR at depth d on the centreline of a circular field of radius r can be described by SPR = aZ + bZ2, with Z = rd/(r + d). The proportionality constant a is determined by the linear attenuation coefficient mu and the ratio r/d. The coefficient b for the second-order term depends on r/d and energy, the latter by being proportional to mu3. This relation between SPR and Z can facilitate analysis of beam data in terms of the contributions from primary and scattered photons, in particular for small depths and small fields where measurements are difficult. This is illustrated by examining measured TAR data for 60Co gamma beams. While the relation between SPR and Z is not fully explained, it is demonstrated qualitatively that SPR approximately equal to aZ if only first-order scatter is to be accounted for.

Humans↗
PubMed1974

Isolation and function of the gene A initiator of bacteriophage phi-chi 174, a highly specific DNA endonuclease.

The gene A product of Escherichia coli bacteriophage varphiX 174, necessary for initiation of varphiX DNA synthesis, has been identified and purified to near homogeneity. The gene A protein is shown to be a highly specific DNA endonuclease which breaks one phosphodiester bond in the viral strand of either double-stranded, circular varphiX replicative form I (RFI), or in single-stranded, circular varphiX viral DNA; it is inactive against all other species of DNA tested.

Carbon Radioisotopes↗
PubMed1992

Affinity purification and characterization of myristoylated alanine-rich protein kinase C substrate (MARCKS) from bovine brain. Comparison of the cytoplasmic and the membrane-bound forms.

A major in vivo substrate of Ca(2+)-phospholipid-dependent protein kinase (myristoylated alanine-rich C-kinase substrate (MARCKS)) has been purified to apparent homogeneity from the particulate as well as from the cytoplasmic fractions of calf brain using a calmodulin affinity column. The two preparations were characterized and compared with various biochemical and biophysical techniques. Although they behave similarly in various chromatographic procedures during purification, their elution positions from the gel filtration column are markedly different. Stokes radii of 85 and 45 A were measured for the cytoplasmic and membrane MARCKS, respectively. Once purified, however, they show a similar small Stokes radius (45 A), suggesting the dissociation of a component or a drastic conformational change in the cytoplasmic preparation during purification. The electrospray mass spectroscopic analysis of the two preparations revealed the existence of at least three major subpopulations with molecular mass differences of 80 daltons, which suggests the presence of protein phosphorylated in different degrees. The cytoplasmic preparation contains more phosphorylated species compared with the membrane preparation, whereas the calculated molecular weight of each peak was indistinguishable between the two preparations. Correspondingly, when the two preparations were phosphorylated by purified protein kinase C in vitro, more phosphate groups were transferred to the membrane preparation (4 mol/mol) than to the cytoplasmic preparation (2.9 mol/mol). A significant difference was also observed in the inhibition of calmodulin of the phosphorylation reaction. On the other hand, the circular dichroism of the two preparations showed similar spectra rich in random coil with little contribution of alpha-helix (approximately 10%), suggesting that there is not a significant difference in the overall conformation. These results clearly established that the two preparations are the same protein coded by a single gene but they differ in their degree of phosphorylation, and that the difference observed in their Stokes radius is due to the presence of an unidentified factor that is removed from the cytoplasmic MARCKS during purification.

Animals↗
PubMedDate not supplied

The putative mitochondrial genome of Plasmodium falciparum.

Intraerythrocytic stages of mammalian malarial parasites employ glycolysis for energy production but some aspects of mitochondrial function appear crucial to their survival since inhibitors of mitochondrial protein synthesis and electron transport have antimalarial effects. Investigations of the putative mitochondrial genome of Plasmodium falciparum have detected organellar rRNAs and tRNAs encoded by a 35 kb circular DNA. Some features of the organization and sequence of the rRNA genes are reminiscent of chloroplast DNAs. The 35 kb DNA also encodes open reading frames for proteins normally found in chloroplast but not mitochondrial genomes. An apparently unrelated 6 kb tandemly repeated element which encodes two mitochondrial protein coding genes and fragments of rRNA genes is also found in malarial parasites. The malarial mitochondrial genome thus appears quite unusual. Further investigations are expected to provide insights into the possible functional relationships between these molecules and perhaps their evolutionary history.

Animals↗
PubMed1981

Regulatory and coding potential of the mouse mammary tumor virus long terminal redundancy.

Molecular clones containing the 3' half of newly integrated mouse mammary tumor virus (MMTV) DNA with adjacent mouse cellular sequences were characterized. In addition, we cloned the long terminal redundancy joint from the unintegrated circular form of MMTV DNA. The entire nucleotide sequence of the integrated and part of the unintegrated terminal redundancy was determined; this allowed us to delineate the boundaries of the MMTV long terminal redundancy, which comprises 1,327 base pairs. The position of possible RNA polymerase II initiation and termination signals corresponded closely to the expected regions of viral RNA initiation and termination specified by current models. The MMTV long terminal redundancy also contained a large open reading frame with sufficient information for a protein of 198 amino acids. Initial comparison of flanking 3' cellular sequences from three independent integrated clones suggested there was no host sequence specificity in the MMTV integration event. However, specificity of integration with respect to viral sequences was precise.

Bacteriophage lambda↗
PubMed1984

[Formation of plasmid pBR322 oligomers as depending on a tetracycline concentration].

Escherichia coli K12 strains containing the plasmid pBR322 often show varying contents of plasmid oligomers, in which the monomer units are arranged in tandem. When the concentration of the plasmid-selective antibiotic tetracycline in the medium becomes increased selection of cells containing largely higher oligomers occurs. The number of monomer units organized in the oligomers increases with tetracycline concentration. recA- mutants are unable to generate oligomers under the same conditions and show lower tetracycline resistance. This observations suggest a selective advantage of oligomer containing cells in the presence of tetracycline as a result of higher gene dosage. But E. coli cells transformed with monomers, dimers, trimers, as well as tetramers of pBR322 are characterized by roughly the same plasmid DNA content as well as plasmid coded beta-lactamase and resistance to tetracycline.

Culture Media↗
PubMed1980

Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.

A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the beta-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA. Second, a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the "signal" sequence thought to be involved in secretion of beta-lactamase.

Base Sequence↗
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↗
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↗