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M Cotten

Publications and source records attributed to M Cotten.

At least 73 records · Page 4Linked to original sources
PubMed1992

Amphibian oocytes and sphere organelles: are the U snRNA genes amplified?

The sphere organelles (spheres) of Xenopus and other amphibian oocytes are known to contain small nuclear ribonucleoprotein particles (snRNPs) and have been suggested to play a role in snRNP complex assembly. Coupled with the similarities that exist between spheres and nucleoli and the quantitative and kinetic aspects of snRNA synthesis in the Xenopus oocyte, we have investigated whether or not the U snRNA encoding genes are amplified in Xenopus oogenesis, the spheres being possible sites for the location of such extrachromosomal gene copies. By applying a number of quantitative nucleic acid hybridization procedures to both total and fractionated oocyte and somatic DNA, employing both homologous and heterologous U snRNA gene probes and suitable amplification and non-amplification control probes, we show that the U snRNA genes do not undergo any major amplification in Xenopus oogenesis. Therefore, the analogy between the sphere organelles and nucleoli appears to be limited. The role of the spheres and their relationship to other snRNP containing structures, specifically B snurposomes, and the sphere organizer loci remains obscure.

Animals↗
PubMed1992

High-efficiency gene transfer mediated by adenovirus coupled to DNA-polylysine complexes.

Employment of recombinant viruses as gene transfer vectors is limited by constraints on the size and functional design of the genetic material to be transferred as well as potential safety hazards deriving from obligatory co-transfer of viral genetic elements. As an alternative strategy that capitalizes on the efficient cellular entry mechanisms of viruses, we have derived adenovirus-polylysine-DNA complexes whereby foreign DNA is transferred bound to the exterior of the virion. This linkage was accomplished utilizing an antibody bridge in which a monoclonal antibody was rendered competent to carry DNA by the attachment of a polylysine residue. Attachment of the antibody-polylysine to the virus was by virtue of the antibody's specificity for the virion. The resulting vector system mediates high-efficiency gene transfer to target cells in vitro. In addition, this vector design allows greatly enhanced flexibility in terms of the size and design of heterologous sequences that can be transferred. Since this strategy selectively exploits viral entry functions, which are independent of viral gene expression, the potential exists to derive vectors that avoid the hazards deriving from transfer of parent virus genome.

Adenoviridae↗
PubMed1992

Gene transfer to respiratory epithelial cells via the receptor-mediated endocytosis pathway.

Gene-based therapies for a variety of inherited and acquired pulmonary diseases will require the development of vectors capable of safe and efficient transfer of DNA to the respiratory epithelium. The present study examined the feasibility of delivering DNA to respiratory epithelial cells by the receptor-mediated endocytosis pathway. This strategy employs molecular conjugates consisting of a cognate moiety, in this case human transferrin, covalently linked to a DNA-binding moiety, such as a cationic polyamine. Complexes were formed between transferrin-polylysine conjugates (hTfpL) and plasmid DNA carrying the firefly luciferase reporter gene (pRSVL). The conjugate-DNA complexes were added directly to cells in tissue culture and incubated for 24 h, after which cell lysates were analyzed for luciferase enzyme activity by luminometry. An immortalized human respiratory epithelial cell line (HBE1) treated with the transferrin-polylysine-DNA complexes exhibited luciferase enzyme activity significantly augmented over background levels. This respiratory epithelial cell line exhibited greater susceptibility to gene transfer by the transferrin-polylysine conjugates than did non-respiratory epithelial cell lines known to possess high levels of transferrin receptors. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the conjugates by the transferrin pathway was verified by competition for the transferrin receptor. In addition, treatment with agents that either increased transferrin receptor number or decreased lysosomal degradation markedly augmented gene expression mediated by the conjugates. Thus, respiratory epithelial cells possess receptors for transferrin that can be exploited to accomplish gene transfer by the receptor-mediated endocytosis pathway.

Animals↗
PubMed1991

A simple procedure for the preparation of protected 2'-O-methyl or 2'-O-ethyl ribonucleoside-3'-O-phosphoramidites.

Protected 2'-O-methyl and 2'-O-ethyl ribonucleoside-3'-O-phosphoramidites were prepared via alkylation of the ribonucleosides at an early stage in the synthesis. Utilizing a strategy of minimal protection, the alkylation was performed with unprotected cytidine and adenosine, or with O6-protected guanosine and N3,5'-O-protected uridine using methyl or ethyl iodide and sodium hydride. In subsequent steps, the introduction of standard protective groups for oligonucleotide synthesis and the concomitant separation from 3'-O-alkylated isomers was accomplished. A modification of the phosphitylation procedure permitted facile isolation of the desired phosphoramidites which show high coupling efficiencies in oligomer assembly.

Ethane↗
PubMed1991

Adenovirus enhancement of transferrin-polylysine-mediated gene delivery.

Gene transfer may be accomplished by the receptor-mediated endocytosis pathway using transferrin-polylysine conjugates. For some target cells, however, gene transfer by this vector is extremely limited, despite the presence of the appropriate surface receptors, a phenomenon attributed to lysosomal degradation of endosome-internalized conjugate-DNA complexes. To enhance DNA escape from the cell vesicle system and thus augment gene transfer by this route, we have used the capacity of adenoviruses to disrupt endosomes as part of their entry mechanism. Adenoviral infection augmented levels of gene transfer by transferrin-polylysine conjugates in a dose-dependent manner: levels of gene transfer of greater than 2000-fold above baseline were achieved. Use of the adenovirus in this context allowed enhanced levels of gene transfer in a variety of target cells, including cell lines otherwise refractory to gene transfer by transferrin-polylysine conjugates. This augmentation was based on adenoviral-mediated vesicle disruption, a process independent of viral gene expression. Thus, the development of specific mechanisms to effect release from the endosome in combination with gene transfer by the receptor-mediated endocytosis pathway will increase the utility of this delivery system by allowing high levels of gene expression in target cells.

Adenoviridae↗
PubMed1991

2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event.

We describe the synthesis of 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides and demonstrate their utility as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. These 2'-O-modified compounds were designed to possess the binding affinity of an RNA molecule towards a complementary RNA target with an enhanced stability against nucleases. The 2'-O-methyl and 2'-O-ethyl antisense compounds function as potent inhibitors of the reaction at 1-10 nM, approximately 5-fold more effective than a natural antisense RNA molecule and requiring an approximate 5-fold excess over the target RNA for 80% inhibition of the processing reaction.

Base Sequence↗
PubMed1991

Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells.

We have previously described a gene delivery system based upon the receptor-mediated endocytosis of DNA complexed with transferrin-polycation conjugates. This delivery system has been found to be very effective for both the internalization and the expression of genetic material in cells that have many transferrin receptors. Upon scrutinization of the parameters involved in this method, which we have termed transferrinfection, we note two important features of the process: the polycation in polycation-transferrin conjugates, as expected, serves to attach the transferrin moiety to the DNA and, in addition, the polycation functions to condense the DNA into a doughnut structure. Electron microscopic analysis of a range of poorly active to highly active transferrinfection samples reveals a strong correlation between DNA condensation and cellular DNA uptake. Furthermore, we demonstrate that the transfection activity of the DNA complex can be increased by addition of free polycation as long as a sufficient quantity of polycation-transferrin conjugates remains in the complex to ensure its binding to the cellular receptor.

Amino Acid Sequence↗
PubMed1990

Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells.

Most current gene transfer methods function satisfactorily in specialized systems involving established cell lines but are often not applicable with nonadherent, primary hematopoietic cells, which are notoriously difficult to transfect. To approach this problem, we have investigated an alternative method of gene transfer, "transferrinfection," in which DNA complexed to transferrin-polycation conjugates is introduced into cells by receptor-mediated endocytosis [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We show here that transferrin-polylysine and transferrin-protamine, when complexed to plasmid DNA containing a luciferase reporter gene, is efficiently bound and moved into avian erythroblasts by endocytosis. Successful transfer and expression of the luciferase reporter gene depends on specific interaction of the transferrin-polylysine-DNA complex with the transferrin receptor and occurs in a significant fraction (greater than 95%) of the cells. Gene transfer efficiency by transferrinfection is lower than with an optimized DEAE-dextran transfection method but reaches similar efficiencies when the cells are treated with chloroquine. Because the procedure in the absence of chloroquine is completely nontoxic to cells, a constant expression level of transferred genes may be maintained by repeated additions of transferrin-polylysine-DNA complex. In addition, the usefulness of transferrinfection for gene transfer into primary hematopoietic cells is demonstrated.

Animals↗
PubMed1990

Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels.

We have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, we have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We demonstrate here that this procedure is useful for a human leukemic cell line. We enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell-permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, we show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks "transferrinfection," as does incubation of the cells at 18 degrees C.

Chloroquine↗
PubMed1990

Transferrin-polycation conjugates as carriers for DNA uptake into cells.

We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection."

Animals↗
PubMed1989

Ribozyme mediated destruction of RNA in vivo.

Previous studies have demonstrated that high ribozyme to substrate ratios are required for ribozyme inhibitory function in nuclear extracts. To obtain high intracellular levels of ribozymes, tRNA genes, known to be highly expressed in most tissues, have been modified for use as ribozyme expression cassettes. Ribozyme coding sequences were placed between the A and the B box, internal promoter sequences of a Xenopus tRNAMet gene. When injected into the nucleus of frog oocytes, the ribozyme tRNA gene (ribtDNA) produces 'hammerhead' ribozymes which cleave the 5' sequences of U7snRNA, its target substrate, with high efficiency in vitro. Oocytes were coinjected with ribtDNA, U7snRNA and control substrate RNA devoid of a cleavage sequence. It was found that the ribtRNA remained localized mainly in the nucleus, whereas the substrate and the control RNA exited rapidly into the cytoplasm. However, sufficient ribtRNA migrated into the cytoplasm to cleave, and destroy, the U7snRNA. Thus, the action of targeted 'hammerhead' ribozymes in vivo is demonstrated.

Animals↗
PubMed1989

Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro.

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

Animals↗
PubMed1988

Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 3' RNA processing reaction.

We have made a detailed molecular analysis of the reactions leading to the formation of mature 3' ends in mammalian histone mRNAs. Using two analytical protocols we have identified an essential sequence motif in the downstream spacer which is consistently present, albeit in diffuse form, mammalian histone genes. Tampering with this sequence element completely abolishes 3' processing. However, 3' cleavage in vitro, although at a very much reduced rate, can be detected when the conserved hairpin is deleted from histone precursor mRNAs. U7 snRNA, previously shown to be essential for the maturation of sea urchin histone messages, was isolated from murine cells and the sequence was determined. The approximately 63-nucleotide, trimethyl-G-capped, murine U7 snRNA possesses a sequence shown in the sea urchin U7 to be required for Sm-precipitability, and like the sea urchin U7, the 3' end of murine U7 is encased in a hairpin structure. The 5' sequence of murine U7 exhibits extensive sequence complementarity to the conserved downstream motif of the histone precursor. As expected, oligo-nucleotide-directed RNase H cleavage of this portion of murine U7 inhibits the in vitro processing reaction. These experiments identify a set of specific contacts between mammalian U7 and histone precursor RNA which is indispensable for the maturation reaction.

Animals↗
PubMed1987

Purification of a novel, nucleoplasmin-like protein from somatic nuclei.

We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S.

Amino Acids↗
PubMed1986

Novobiocin inhibits passive chromatin assembly in vitro.

Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type II topoisomerase enzymes, interferes with in vitro chromatin assembly using purified histones, DNA and nucleoplasmin. The target of inhibition is not topoisomerase II; this energy-independent assembly system lacks any ATP and Mg2+-dependent type II topoisomerase or gyrase activities. Rather, novobiocin interacts with histones, disrupting histone-histone associations required for octamer formation, and causing histones to precipitate from both nucleoplasmin-histone and histone-DNA complexes. Thus, novobiocin is able to generate 'dynamic' chromatin in vitro in the absence of ATP and Mg2+ by removing histones from previously assembled static chromatin, so that the DNA supercoils, previously constrained by conventional nucleosomes, become susceptible to removal by topoisomerase I.

Animals↗
PubMed1986

Massive phosphorylation distinguishes Xenopus laevis nucleoplasmin isolated from oocytes or unfertilized eggs.

Nucleoplasmin isolated from unfertilized Xenopus laevis eggs possesses an in vitro chromatin assembly activity which is superior to nucleoplasmin isolated from oocytes. It is demonstrated here that the two forms of the protein differ in the amount of attached phosphate, with the egg protein possessing nearly 20 phosphate groups per protein monomer and the oocyte protein possessing less than 10 phosphate groups per monomer. A kinase preparation from unfertilized eggs is shown to be capable of modifying oocyte nucleoplasmin so that it displays the electrophoretic heterogeneity of egg nucleoplasmin. Furthermore, when the egg protein is treated with phosphatase and repurified, the chromatin assembly activity deteriorates to the level of the oocyte protein.

Animals↗