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PubMed2000

Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and primary lymphoma cells can be efficiently transfected using an adenoviral vector system. By adding cationic liposomes, the efficiency of adenoviral gene transfer to primary tumor cells could be further improved. This protocol may have an impact on the use of lymphoma cells in cancer gene therapy.

Adenoviridae↗

PubMed1999

Efficient adventitial gene delivery to rabbit carotid artery with cationic polymer-plasmid complexes.

Different lipids and cationic polymers were tested in vitro for their ability to transfect rabbit aortic smooth muscle cells and human endothelial cells with lacZ marker gene. Toxicity of the complexes was evaluated with MTT assay. Selected plasmid-polymer complexes with different charge ratios were then tested for in vivo gene transfer efficiency using adventitial gene transfer by placing a silastic gene delivery reservoir (collar) around the carotid artery. Transfection efficiency was determined by X-gal staining 3 days after the gene transfer. Based on in vitro experiments, fractured polyamidoamine dendrimers and polyethylenimines (PEI) were selected for in vivo experiments. Fractured dendrimers (generation 6, +/- charge ratio of 3) had the highest in vivo gene transfer efficiency (4.4% +/- 1.7). PEI with molecular size of 25 kDa (+/- charge ratio 4) was also effective (2.8% +/- 1.8) in this model. PEI of 800 kDa showed a constant but modest gene transfer efficiency (1.8% +/- 0.1) with all charge ratios. A low level gene transfer was also detected with naked DNA (0.5% +/- 0.3). No signs of inflammation were seen in any of the study groups. We show here that in vitro cell culture experiments can be used to identify efficient in vivo gene transfer methods for arterial gene therapy, but the charge ratios for each complex must be optimized in vivo. It is concluded that fractured dendrimer and PEI are efficient gene delivery vehicles and can be used for arterial gene therapy via adventitial gene delivery route.

Animals↗

PubMed2000

Efficient and sustained transgene expression in human corneal cells mediated by a lentiviral vector.

The development of vectors and techniques able to transfer potentially therapeutic genetic information to corneal tissues efficiently may have broad clinical applications. Although a variety of vectors have been tested for their ability to transduce corneal tissue, these systems have been ineffective at transducing all cell types or have been associated with a relatively short duration of transgene expression. Towards the implementation of efficient, long-term transgene expression in all corneal cell types, we have studied the ability of a recombinant lentiviral vector, containing the enhanced green fluorescent protein (eGFP), to mediate gene transfer into human corneal tissue in vitro and in situ. Human primary keratocytes, cultured in vitro, were efficiently transduced by a lentiviral vector as determined by fluorescent-activated cell sorting (FACS) and by fluorescent microscopy. Transduction efficiency was found to be dependent upon multiplicity of infection (MOI); 92% of keratocytes were transduced at an MOI of 1000. The proportion of eGFP-positive cells remained unchanged throughout continuous culture for 60 days, indicating stable expression and a lack of selective pressure for or against transduced cells. Human corneal tissue, obtained at the time of penetrating keratoplasty, demonstrated efficient in situ transduction with this vector. Endothelial cells, epithelial cells and stromal keratocytes at the exposed cut edge of the corneal tissue in situ demonstrated eGFP expression. Underlying stromal cells not in direct contact with vector-containing media, were not transduced, implying that virus-cell contact is required for transduction. Transduced corneal tissues expressed eGFP in situ for the life of the corneal button in extended organ culture (60 days). These results imply that lentiviral vectors may prove to be useful tools, able to transduce corneal tissue efficiently, and that transgene expression is temporally stable. Gene Therapy (2000) 7, 196-200.

Cells, Cultured↗

PubMed2000

Co-polymer of histidine and lysine markedly enhances transfection efficiency of liposomes.

Development of nonviral delivery systems is progressing toward a transfection efficiency sufficient to affect metabolic and neoplastic diseases in humans. Nevertheless, inadequate transfection efficiency of target cells with current nonviral systems still limits the utility of this therapy. In the current study, we have determined that a co-polymer of histidine and lysine (H-K) enhances the transfection efficiency of liposomes, a leading nonviral system. We found that in the absence of serum, the addition of this polymer increased transfection as much as 10-fold in comparison with the liposome:DNA complex alone. More impressively, the co-polymer in the presence of serum increased transfection efficiency up to 100-fold. Furthermore, in vivo expression of luciferase in a tumor increased 15-fold with the addition of H-K polymer to the liposome:plasmid DNA complexes. Without liposomes, the H-K polymer had little to no effect on transfection efficiency. We anticipate that further modifications of this co-polymer will yield molecules with both increased complexity and transfection efficiency.

3T3 Cells↗

PubMed2002

Targeting of cell receptors and gene transfer efficiency: a balancing act.

Vectors conjugated with ligands recognized by cell surface receptors are of interest for cystic fibrosis gene therapy since these vectors would allow cell-specific targeting. However, an efficient and specific uptake may be abrogated by a subsequent intracellular trafficking leading to an inefficient gene transfer. This has been shown for polylysine substituted with mannose residues. While mannose-specific membrane lectins are predominantly expressed at the surface of airway cells and mannosylated complexes are the most efficiently incorporated glycosylated complexes in these cells, mannosylated complexes lead to a low gene transfer efficiency because of an inefficient exit from endosomal compartments, a high accumulation in lysosomes and an inefficient nuclear import. In contrast, the entry of low amounts of lactosylated complexes is balanced by more efficient intracellular trafficking, leading to an efficient gene transfer. This emphasizes that for a successful gene transfer, it is necessary to find the balance between efficient and specific uptake, and intracellular trafficking that overcomes the various cellular barriers and enables the plasmid to reach the nucleus.

Biological Transport↗

PubMed2003

High-efficiency plasmid gene transfer into dystrophic muscle.

The efficiency of plasmid gene transfer in skeletal muscle is significantly enhanced by pretreatment with hyaluronidase and the application of an electrical field to the muscle following the injection of plasmid DNA, a process referred to as electrotransfer. However, the presence of increased levels of connective tissue in muscular dystrophies, such as Duchenne muscular dystrophy (DMD), may affect the efficiency of this process. Here we demonstrate that the efficiency of electrotransfer is not affected by increased levels of connective tissue in the mdx mouse model of DMD and that any damage induced by the electrotransfer process is not exacerbated in the dystrophic phenotype. However, increasing the concentration of hyaluronidase does not improve transfection efficiencies further. Unlike direct injection of plasmid DNA, the efficiency of electrotransfer is not dependent upon the sex and age of mice used. The combined treatment of hyaluronidase and electrotransfer results in highly efficient gene transfer in dystrophic muscle with limited muscle damage.

Animals↗

PubMed2005

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities.

Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1alpha promoter (EF1alpha promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/beta-actin promoter with beta-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+ CD38(low/-) and CD34+ AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

Actins↗

PubMed2005

Aspiration and sampling efficiencies of the TSP and louvered particulate matter inlets.

An experimental system was developed for the rapid measurement of the aspiration/transfer efficiency of aerosol samplers in a wind tunnel. We attempted to measure the aspiration and particle transfer characteristics of two inlets commonly used for sampling airborne Particulate Matter (PM): the 'Total Suspended Particulate' or TSP inlet, and the louvered 'dichotomous sampler inlet' typically used in sampling PM10 or PM2.5. We were able to determine the fraction of the external aerosol that enters the inlet and is transferred through it, and hence is available for collection by a filter, or further size fractionation into PM10 or PM2.5. This 'sampling efficiency' was analysed as a function of dimensionless aerodynamic parameters in order to understand the factors governing inlet performance. We found that for the louvered inlet the sampling efficiency increases as the external wind increases. Under all conditions expected in practical use the louvered inlet aspirates sufficient PM to allow either PM10 or PM2.5 to be selected downstream. The TSP inlet's sampling efficiency decreases with increasing external wind, and the TSP inlet is likely to under-sample the coarse end of the PM10 fraction at moderate and high external winds. As this inlet is generally not used with a downstream size fractionator, changes in sampling efficiency directly affect the measured aerosol concentration. We also investigated whether it is possible to dimensionally scale the PM inlets to operate at either higher or lower flow rates, while preserving the same sampling characteristics as the current full-scale, 16.67 L min(-1) versions. In the case of the louvered inlet, our results indicate that scaling to lower flow rates is possible; scaling to higher flow rates was not tested. For the TSP sampler, the sampling efficiency changes if the sampler is scaled to operate at smaller or larger flow rates, leading to unreliable performance.

Aerosols↗

PubMed2005

Model studies of the (6-4) photoproduct photoreactivation: efficient photosensitized splitting of thymine oxetane units by covalently linked tryptophan in high polarity solvents.

Three covalently linked tryptophan-thymine oxetane compounds used as a model of the (6-4) photolyase-substrate complex have been prepared. Under 290 nm light, efficient splitting of the thymine oxetane with aromatic carbonyl compounds gives the thymine monomer and the corresponding carbonyl compounds by the covalently linked tryptophan via an intramolecular electron transfer, and exhibits a strong solvent dependence: the quantum yield (Phi) is ca. 0.1 in dioxane, and near 0.3 in water. Electron transfer from the excited tryptophan residue to the oxetane unit is the origin of fluorescence quenching of the tryptophan residue, and is more efficient in strong polar solvents. The splitting efficiency of the oxetane radical anion within the tryptophan.+-oxetane.- species is also solvent-dependent, ranging from ca. 0.2 in dioxane to near 0.35 in water. Thus, the back electron transfer reaction in the charge-separated species would be suppressed in water, but is still a main factor causing low splitting efficiencies in the tryptophan-oxetane systems. In contrast to the tryptophan-oxetane system, fast nonradiation processes are the main causes of low efficiency in the flavin-oxetane system. Hence, nonradiative processes of the excited FADH-, rather than electron transfer to oxetane, may be an important factor for the low repair efficiency of (6-4) photolyase.

Electrons↗

PubMed2002

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling.

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.

Adenylyl Cyclases↗

PubMed2002

Muscle fibre type, efficiency, and mechanical optima affect freely chosen pedal rate during cycling.

UNLABELLED: This study investigated the variation in freely chosen pedal rate between subjects and its possible dependence on percentage myosin heavy chain I (%MHC I) in m. vastus lateralis, maximum leg strength and power, as well as efficiency. Additionally, the hypothesis was tested that a positive correlation exists between percentage MHC I and efficiency at pre-set pedal rates but not at freely chosen pedal rate. Twenty males performed cycling at low and high submaximal power output ( approximately 40 and 70% of the power output at which maximum oxygen uptake (VO(2max)) was attained at 80 r.p.m.) with freely chosen and pre-set pedal rates (61, 88, and 115 r.p.m.). Percentage MHC I as well as leg strength and power were determined. Freely chosen pedal rate varied considerably between subjects: 56-88 r.p.m. at low and 61-102 r.p.m. at high submaximal power output. This variation was only partly explained by percentage MHC I (21-97%) as well as by leg strength and power. Interestingly, %MHC I correlated significantly with the pedal rate at which maximum peak crank power occurred (r = -0.81). As hypothesized, %MHC I and efficiency were unrelated at freely chosen pedal rate, which was in contrast to a significant correlation found at pre-set pedal rates (r = 0.61 and r = 0.57 at low and high power output, respectively). CONCLUSIONS: Subjects with high percentage MHC I chose high pedal rates close to the pedal rates at which maximum peak crank power occurred, while subjects with low percentage MHC I tended to choose lower pedal rates, favouring high efficiency. Nevertheless, the considerable variation in freely chosen pedal rate between subjects was neither fully accounted for by percentage MHC I nor by leg strength and power. Previously recognized relationships between percentage Type I ( approximately %MHC I) and efficiency as well as between pedal rate and efficiency were confirmed for pre-set pedal rates, but for freely chosen pedal rate, these variables were unrelated.

Adolescent↗

PubMed1996

The Mu strong gyrase-binding site promotes efficient synapsis of the prophage termini.

A strong DNA gyrase-binding site (SGS) is located midway between the termini of the bacteriophage Mu genome and is required for efficient replicative transposition. We have proposed that the SGS promotes the efficient synapsis of the Mu prophage ends (an obligate early step in replicative transposition), and that it does so by helping to organize the prophage DNA into a supercoiled loop with the SGS at the apex of the loop and the prophage termini at the base. The positioning of the synapsing termini equidistant from the SGS is a key element in the proposed model. To test this proposal, we have constructed prophages with a second, internal right end and asked whether the natural, external right end or the internal right end is used for synapsis with the left end in the presence and absence of the SGS. In the presence of the central SGS, the natural, or outside, right end was used exclusively and very efficiently. In the absence of the central SGS, the internal right end was used preferentially and inefficiently: the efficiency of transposition decreased with increasing distance between the internal right end and the left end. Repositioning the SGS midway between the left end and an internal right end allowed highly efficient use of the internal right end. These results support a model in which gyrase can influence long-range DNA interactions to promote efficient synapsis of Mu prophage ends.

Bacteriolysis↗

PubMed1994

Structural features of myelin basic protein mRNAs influence their translational efficiencies.

The myelin basic protein (MBP) gene expresses several alternatively spliced products with the same 5' and 3' untranslated regions (UTRs). It has been reported that its expression may be regulated not only at the transcriptional level but also at the translational level during development. We engineered several MBP mRNA deletion mutants with 5' (-48, -37, -27, -22, and -10) and 3' UTRs of differing lengths and examined the translational efficiencies of these constructs in cell-free systems. The translational efficiencies of the constructs differed significantly over a range of almost 10-fold. A deletion of 11 nucleotides from the 5' end of the natural (i.e., -48) MBP mRNA resulted in an approximate fourfold reduction in translational efficiency. Further truncation of the 5' UTR increased the translational efficiencies of the constructs as has been observed with many RNAs. These results suggest that there may be a positive control element between -48 and -37 nucleotides in the 5' UTR of MBP mRNA. The effects of modifying the lengths of the 5' UTR on the translational efficiency of mRNAs encoding the 21.5-kDa and 14-kDa MBPs were the same, suggesting that the effect observed was not unique to the 21.5-kDa MBP mRNA. Truncating the 3' UTR of four different alternatively spliced MBP mRNAs also altered their translational efficiencies. Thus, the 5' and 3' UTRs of MBP mRNAs appear to influence the translation of these mRNAs, and such factors may be involved in the translational regulation of MBP gene expression.

Animals↗

PubMed2001

Depressive symptoms and history of depression predict rehabilitation efficiency in stroke patients.

OBJECTIVE: To examine how depressive symptoms, a history of depression, and cognitive functioning contribute to the prediction of rehabilitation efficiency in stroke patients. DESIGN: Consecutive admissions to an acute inpatient rehabilitation program were screened for cognitive functioning and level of depressive symptoms. History of depression was determined by family member interview. Functional status was evaluated at time of admission and discharge. Depressive symptoms, depression history, and cognitive functioning were examined as predictors of length of stay (LOS) and efficiency of utilization of rehabilitation services. SETTING: Acute inpatient rehabilitation hospital. PATIENTS: A total of 348 consecutive stroke admissions to an inpatient program were evaluated for depression and cognitive functioning, of whom 243 patients completed all aspects of the screening. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Rehabilitation progress, measured with the LOS efficiency measure (LOS-EFF) of the FIM instrument, and length of rehabilitation hospital stay. RESULTS: Patients with higher levels of depressive symptoms used rehabilitation services less efficiently than those with lower symptom levels but did not have longer LOSs. History of depression was associated with longer LOS and less efficient use of rehabilitation services. Cognitive impairment did not predict rehabilitation efficiency. CONCLUSIONS: The present study lends further support to the benefits of screening stroke patients at the time of rehabilitation admission for depression and history of depression. Identifying patients who have high levels of depressive symptoms and/or a previous depressive episode will allow more comprehensive assessment and rapid intervention.

Aged↗

PubMed2002

Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications.

The mechanisms that link genotypes of hepatitis D virus (HDV) with clinical outcomes have not yet been elucidated. Genotypic variations are unevenly distributed along the sequences of hepatitis delta antigens (HDAgs). Of these variations, the packaging signal at the C-terminus has a divergence of 74% between genotypes I and II. In this report, we address the issue of whether these high variations between genotypes affect assembly efficiency of HDV particles and editing efficiency of RNA. Viral package systems of transfection with expression plasmids of hepatitis B surface antigen and HDAgs or whole genomes of HDV consistently indicate that the package efficiency of genotype I HDV is higher than that of genotype II. Segment swapping of large-form HDAg indicates that the C-terminal 19-residue region plays a key role for the varied assembly efficiencies. Also, the editing efficiency of genotype I HDV is higher than that of genotype II. The nucleotide and structural changes surrounding the editing site may explain why genotype II HDV has a low RNA editing efficiency. The findings of in vitro assembly systems were further supported by the observations that patients infected with genotype II had significantly lower alanine transaminase (ALT) levels, more favorable outcomes (P <.05), and a trend to have lower serum HDV RNA levels as compared with those infected with genotype I HDV (P =.094). In conclusion, genotype II HDV secretes fewer viral particles than genotype I HDV does, which in turn may reduce the extent of infection of hepatocytes and result in less severe hepatic inflammation.

Adult↗

PubMed1996

Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.

Animals↗

PubMed1995

Efficient plasmid DNA replication in Xenopus egg extracts does not depend on prior chromatin assembly.

Small plasmids replicate efficiently in unfertilized Xenopus eggs provided they are injected before rather than after activation of the cell cycle. Here we use Xenopus egg extracts to test the hypothesis that efficient replication results from chromatin assembly prior to activation giving preloaded plasmids a head start toward the formation of a replicating pseudonucleus (Sanchez, J.A., Marek, D., and Wangh, L.J. (1992) J. Cell Sci. 103, 907-918). As in ovum, plasmid DNA preincubated in unactivated egg cytoplasmcytostatic factor extracts) replicate more efficiently after extract activation than does the same DNA added to the same extract after activation. Unlike in ovum, however, plasmids that replicate efficiently in vitro do not assemble into chromatin during preincubation and become topologically knotted instead. But even DNA knotting does not explain subsequent efficient replication. Also, plasmids preassembled into chromatin in vitro do not replicate efficiently in activated egg cytoplasm unless first preincubated in a CSF extract. We conclude that unactivated eggs contain replication-enhancing activities that can act independently of plasmid chromatin assembly and DNA topology. These postulated "preloading" factor(s) may be related to licensing factor, an activity that controls initiation of DNA replication in eukaryotic cells. The experimental conditions described here will permit characterization of preloading/licensing factor(s) in the context of a small plasmid substrate.

Animals↗

PubMed1998

Two efficiency elements flanking the editing site of cytidine 6666 in the apolipoprotein B mRNA support mooring-dependent editing.

Normally, apolipoprotein B (apoB) mRNA editing deaminates a single cytidine (C6666) in apoB mRNA. However, when the catalytic subunit of the editing enzyme complex, APOBEC-1, was overexpressed in transgenic mice and rabbits, numerous cytidines in the apoB mRNA and in a novel mRNA, NAT1, were aberrantly hyperedited, and the animals developed liver dysplasia and hepatocellular carcinomas. To identify the RNA motifs in the apoB mRNA that support physiological editing and those that support aberrant hyperediting, we constructed rabbit apoB RNA substrates and tested them in vitro for physiological editing and hyperediting. Three previously unrecognized RNA elements that are critical for efficient physiological editing at C6666 were identified. In concert with the mooring sequence (6671-6681), the 5' efficiency element (6609-6628), an A-rich region (6629-6640), and the 3' efficiency element (6717-6747) increased editing at C6666. The 5' efficiency element was the most potent, elevating physiological editing to wild-type levels in combination with the mooring sequence. The 3' efficiency element was somewhat less important but increased physiological editing to levels approaching wild type. These elements encompass 139 nucleotides on the apoB RNA transcript and are sufficient for editing with the efficiency of full-length apoB mRNA. Furthermore, a distal downstream apoB region (6747-6824) may function as a recognition element in the apoB mRNA. Hyperediting at C6802 in the rabbit apoB mRNA is mediated by RNA elements similar to those required for normal physiological editing at C6666. Similarly sized upstream and downstream flanking regions of C6802 are necessary for hyperediting in combination with a degenerate mooring sequence.

APOBEC-1 Deaminase↗