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1
Vasudevachari, M. B., V. Natarajan, and N. P. Salzman. "Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters." Molecular and Cellular Biology 7, no. 3 (March 1987): 1063–69. http://dx.doi.org/10.1128/mcb.7.3.1063-1069.1987.
Full textAbstract:
Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.
2
Vasudevachari, M. B., V. Natarajan, and N. P. Salzman. "Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters." Molecular and Cellular Biology 7, no. 3 (March 1987): 1063–69. http://dx.doi.org/10.1128/mcb.7.3.1063.
Full textAbstract:
Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.
3
Kaufman, R. J., M. V. Davies, V. K. Pathak, and J. W. Hershey. "The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs." Molecular and Cellular Biology 9, no. 3 (March 1989): 946–58. http://dx.doi.org/10.1128/mcb.9.3.946-958.1989.
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Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.
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Kaufman, R. J., M. V. Davies, V. K. Pathak, and J. W. Hershey. "The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs." Molecular and Cellular Biology 9, no. 3 (March 1989): 946–58. http://dx.doi.org/10.1128/mcb.9.3.946.
Full textAbstract:
Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.
5
Alwine, J. C. "Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins." Molecular and Cellular Biology 5, no. 5 (May 1985): 1034–42. http://dx.doi.org/10.1128/mcb.5.5.1034-1042.1985.
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The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.
6
Alwine, J. C. "Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins." Molecular and Cellular Biology 5, no. 5 (May 1985): 1034–42. http://dx.doi.org/10.1128/mcb.5.5.1034.
Full textAbstract:
The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.
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Chen, XM, SH Zhou, J. Fan, MJ Hu, SQ Wang, and YS Yu. "Construction and Identification of an Antisense Glucose Transporter-1 Plasmid." Journal of International Medical Research 36, no. 5 (October 2008): 1001–7. http://dx.doi.org/10.1177/147323000803600517.
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Our aim was to construct a pcDNA3.1(+) eucaryotic expression system vector containing the antisense glucose transporter-1 ( Glut-1) gene. Total RNA was isolated from human Hep-2 laryngeal carcinoma cells, and the Glut-1 and antisense Glut-1 sequences were amplified by polymerase chain reaction. Expression plasmids containing the sense and antisense cDNA were constructed using the pcDNA3.1(+) vector. The resulting sense and antisense vectors, pcDNA3.1(+)-Glut-1 and pcDNA3.1(+)-antiGlut-1, respectively, were examined by restriction analysis and DNA sequencing. The pcDNA3.1(+)-antiGlut-1 was subsequently transfected into Hep-2 cells. Anti Glut-1 mRNA expression was detected, indicating the successful construction of an antisense Glut-1 plasmid capable of transfecting Hep-2 laryngeal carcinoma cells. These data provide a firm basis for additional studies using the plasmid pcDNA3.1(+)-antiGlut-1 to determine its therapeutic potential for the treatment of laryngeal carcinoma.
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Losos, Jan K., David H. Evans, and Ann M. Verrinder Gibbins. "Targeted modification of the complete chicken lysozyme gene by poxvirus-mediated recombination." Biochemistry and Cell Biology 83, no. 2 (April 1, 2005): 230–38. http://dx.doi.org/10.1139/o05-025.
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We have developed a novel ex vivo system for the rapid one-step targeted modification of large eucaryotic DNA sequences. The highly recombinant environment resulting from infection of rabbit cornea cells with the Shope fibroma virus was exploited to mediate precise modifications of the complete chicken lysozyme gene domain (21.5 kb). Homologous recombination was designed to occur between target DNA (containing the complete lysozyme gene domain) maintained in a λ bacteriophage vector and modified targeting DNA maintained in a plasmid. The targeting plasmids were designed to transfer exogenous sequences (for example, β-galactosidase α-complement, green fluorescent protein, and hydrophobic tail coding sequences) to specific sites within the lysozyme gene domain. Cotransfection of the target phage and a targeting plasmid into Shope fibroma virus infected cells resulted in the poxvirus-mediated transfer of the modified sequences from plasmid to phage. Phage DNA (recombinant and nonrecombinant) was then harvested from the total cellular DNA by packaging into λ phage particles and correct recombinants were identified. Four different gene-targeting pairings were carried out, and from 3% to 11% of the recovered phages were recombinant. Using this poxvirus-mediated targeting system, four different regions of the chicken lysozyme gene domain have been modified precisely by our research group overall with a variety of inserts (6–971 bp), deletions (584–3000 bp), and replacements. We have never failed to obtain the desired recombinant. Poxvirus-mediated recombination thus constitutes a routine, rapid, and remarkably efficient genetic engineering system for the precise modification of large eucaryotic gene domains when compared with traditional practices.Key words: chicken lysozyme, gene targeting, homologous recombination, poxvirus, avian bioreactor.
9
Price, Brian M., Adriane L. Liner, Sukjoon Park, Stephen H. Leppla, Alfred Mateczun, and Darrell R. Galloway. "Protection against Anthrax Lethal Toxin Challenge by Genetic Immunization with a Plasmid Encoding the Lethal Factor Protein." Infection and Immunity 69, no. 7 (July 1, 2001): 4509–15. http://dx.doi.org/10.1128/iai.69.7.4509-4515.2001.
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ABSTRACT The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) ofB. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 μg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.
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Chakrabarti, S., and M. M. Seidman. "Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative." Molecular and Cellular Biology 6, no. 7 (July 1986): 2520–26. http://dx.doi.org/10.1128/mcb.6.7.2520-2526.1986.
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When plasmids carrying a fragmented gene with segments present as direct repeats are introduced into mammalian cells, recombination or gene conversion between the repeated sequences can reconstruct the gene. Intramolecular recombination leads to the deletion of the intervening sequences and the loss of one copy of the repeat. This process is known to be stimulated by double-strand breaks. Two current models for recombination in eucaryotic cells propose that the reaction is initiated by double-strand breaks, but differ in their predictions as to the fate of the intervening sequences. One model suggests that these sequences are always lost, while the other indicates that the reaction will be conservative as a function of the position of the double-strand break. We have constructed a plasmid in which two overlapping portions of the simian virus 40 early region, which contains the origin and T-antigen gene, are present as direct repeats separated by sequences containing a plasmid with a simian virus 40 origin of replication. Recombination across the repeated segments could produce a plasmid with an origin of replication and/or a plasmid with a gene for a functional T-antigen which would drive the replication of both. Introduction of this construction into African green monkey kidney cells, without coinfection, establishes a condition in which the products of the recombination or gene conversion can be interpreted unambiguously. We find that the majority of the reconstruction reactions are nonconservative.
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Chakrabarti, S., and M. M. Seidman. "Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative." Molecular and Cellular Biology 6, no. 7 (July 1986): 2520–26. http://dx.doi.org/10.1128/mcb.6.7.2520.
Full textAbstract:
When plasmids carrying a fragmented gene with segments present as direct repeats are introduced into mammalian cells, recombination or gene conversion between the repeated sequences can reconstruct the gene. Intramolecular recombination leads to the deletion of the intervening sequences and the loss of one copy of the repeat. This process is known to be stimulated by double-strand breaks. Two current models for recombination in eucaryotic cells propose that the reaction is initiated by double-strand breaks, but differ in their predictions as to the fate of the intervening sequences. One model suggests that these sequences are always lost, while the other indicates that the reaction will be conservative as a function of the position of the double-strand break. We have constructed a plasmid in which two overlapping portions of the simian virus 40 early region, which contains the origin and T-antigen gene, are present as direct repeats separated by sequences containing a plasmid with a simian virus 40 origin of replication. Recombination across the repeated segments could produce a plasmid with an origin of replication and/or a plasmid with a gene for a functional T-antigen which would drive the replication of both. Introduction of this construction into African green monkey kidney cells, without coinfection, establishes a condition in which the products of the recombination or gene conversion can be interpreted unambiguously. We find that the majority of the reconstruction reactions are nonconservative.
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Wittekind, M., J. Dodd, L. Vu, J. M. Kolb, J. M. Buhler, A. Sentenac, and M. Nomura. "Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae." Molecular and Cellular Biology 8, no. 10 (October 1988): 3997–4008. http://dx.doi.org/10.1128/mcb.8.10.3997-4008.1988.
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The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon.
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Wittekind, M., J. Dodd, L. Vu, J. M. Kolb, J. M. Buhler, A. Sentenac, and M. Nomura. "Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae." Molecular and Cellular Biology 8, no. 10 (October 1988): 3997–4008. http://dx.doi.org/10.1128/mcb.8.10.3997.
Full textAbstract:
The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon.
14
Clark, C. G., and G. A. Cross. "rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid." Molecular and Cellular Biology 7, no. 9 (September 1987): 3027–31. http://dx.doi.org/10.1128/mcb.7.9.3027-3031.1987.
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An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.
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Clark, C. G., and G. A. Cross. "rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid." Molecular and Cellular Biology 7, no. 9 (September 1987): 3027–31. http://dx.doi.org/10.1128/mcb.7.9.3027.
Full textAbstract:
An extrachromosomal DNA was discovered in Naegleria gruberi. The 3,000 to 5,000 copies per cell of this 14-kilobase-pair circular plasmid carry all the 18S, 28S, and 5.8S rRNA genes. The presence of the ribosomal DNA of an organism exclusively on a circular extrachromosomal element is without precedent, and Naegleria is only the third eucaryotic genus in which a nuclear plasmid DNA has been found.
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Hoffman-Liebermann, B., D. Liebermann, A. Troutt, L. H. Kedes, and S. N. Cohen. "Human homologs of TU transposon sequences: polypurine/polypyrimidine sequence elements that can alter DNA conformation in vitro and in vivo." Molecular and Cellular Biology 6, no. 11 (November 1986): 3632–42. http://dx.doi.org/10.1128/mcb.6.11.3632-3642.1986.
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We previously have shown that homologs of the outer domain segment of the inverted repeat termini (IVR-OD) of the sea urchin TU transposons are conserved among multiple eucaryotic species, including humans. We report here that two cloned human DNA IVR-OD homologs, Hut2 and Hut17, consist of a series of tandem repeats of the trimer AGG/TCC, forming segments (313 and 221 base pairs in length, respectively) of polypurine/polypyrimidine (pPu/pPy or "Puppy") asymmetry in the two DNA strands; these are punctuated at certain sites with variant trimers, which are different for the two clones. Sequences homologous to the Hut2 pPu/pPy tract exist at multiple sites in the DNA of a wide variety of eucaryotes. Hybridization of human DNA with a Hut2 probe or with a previously described chicken DNA pPu/pPy sequence indicates that pPu/pPy sequences can be grouped into families distinguishable by the extent of their homology with each probe at different hybridization stringencies. Moreover, particular pPu/pPy tracts show species-specific differences in their distribution. Both the Hut2 and Hut17 pPu/pPy tracts are cleaved by S1 nuclease when tested on supercoiled plasmids. Most if not all of the 313-base-pair Hut2 pPu/pPy tract is also sensitive to S1 in its native location in HeLa cell chromatin, indicating that the sequence contains conformational information that can be expressed in vivo. This view is supported by evidence that exogenously derived Hut2 pPu/pPy tracts introduced into mouse L cells and integrated in chromatin can assume an S1-sensitive conformation.
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Hoffman-Liebermann, B., D. Liebermann, A. Troutt, L. H. Kedes, and S. N. Cohen. "Human homologs of TU transposon sequences: polypurine/polypyrimidine sequence elements that can alter DNA conformation in vitro and in vivo." Molecular and Cellular Biology 6, no. 11 (November 1986): 3632–42. http://dx.doi.org/10.1128/mcb.6.11.3632.
Full textAbstract:
We previously have shown that homologs of the outer domain segment of the inverted repeat termini (IVR-OD) of the sea urchin TU transposons are conserved among multiple eucaryotic species, including humans. We report here that two cloned human DNA IVR-OD homologs, Hut2 and Hut17, consist of a series of tandem repeats of the trimer AGG/TCC, forming segments (313 and 221 base pairs in length, respectively) of polypurine/polypyrimidine (pPu/pPy or "Puppy") asymmetry in the two DNA strands; these are punctuated at certain sites with variant trimers, which are different for the two clones. Sequences homologous to the Hut2 pPu/pPy tract exist at multiple sites in the DNA of a wide variety of eucaryotes. Hybridization of human DNA with a Hut2 probe or with a previously described chicken DNA pPu/pPy sequence indicates that pPu/pPy sequences can be grouped into families distinguishable by the extent of their homology with each probe at different hybridization stringencies. Moreover, particular pPu/pPy tracts show species-specific differences in their distribution. Both the Hut2 and Hut17 pPu/pPy tracts are cleaved by S1 nuclease when tested on supercoiled plasmids. Most if not all of the 313-base-pair Hut2 pPu/pPy tract is also sensitive to S1 in its native location in HeLa cell chromatin, indicating that the sequence contains conformational information that can be expressed in vivo. This view is supported by evidence that exogenously derived Hut2 pPu/pPy tracts introduced into mouse L cells and integrated in chromatin can assume an S1-sensitive conformation.
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Scafe, C., C. Martin, M. Nonet, S. Podos, S. Okamura, and R. A. Young. "Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits." Molecular and Cellular Biology 10, no. 3 (March 1990): 1270–75. http://dx.doi.org/10.1128/mcb.10.3.1270-1275.1990.
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Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.
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Scafe, C., C. Martin, M. Nonet, S. Podos, S. Okamura, and R. A. Young. "Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits." Molecular and Cellular Biology 10, no. 3 (March 1990): 1270–75. http://dx.doi.org/10.1128/mcb.10.3.1270.
Full textAbstract:
Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.
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Lipp, M., R. Schilling, S. Wiest, G. Laux, and G. W. Bornkamm. "Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene." Molecular and Cellular Biology 7, no. 4 (April 1987): 1393–400. http://dx.doi.org/10.1128/mcb.7.4.1393-1400.1987.
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Recombinant plasmids of the human c-myc promoter-leader region and the bacterial chloramphenicol acetyltransferase (cat) gene were constructed. After transfection into different rodent and human cells, the 862-base-pair (bp) PvuII fragment carrying both c-myc promoters and 350 bp of the untranslated leader conferred 1/15 to 1/30 of the CAT activity mediated by the simian virus 40 promoter. The presence of additional sequences upstream of the PvuII fragment had an overall negative effect on c-myc promoter activity detectable by titration analysis with small amounts of transfected plasmid DNA. The analysis of numerous deletion constructs in the c-myc promoter-leader region as well as S1 mapping experiments demonstrated that the high CAT activity depended largely on the presence of the second promoter. By cotransfection of c-myc-cat constructs with plasmids carrying different parts of the c-myc promoter locus, targets for positively acting cellular factors were identified. Two positive regulatory elements were mapped within the 862-bp PvuII fragment. One was localized within the 248-bp PvuII-SmaI fragment -101 to -349 bp upstream of the first cap site and the other within the 142-pb XhoI-NaeI fragment of the first exon, comprising positions -95 to +47 relative to the second cap site. We conclude that the dual promotor of the human c-myc gene represents a strong eucaryotic promotor regulated by cooperation of positively and negatively acting cellular transcription factors.
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Lipp, M., R. Schilling, S. Wiest, G. Laux, and G. W. Bornkamm. "Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene." Molecular and Cellular Biology 7, no. 4 (April 1987): 1393–400. http://dx.doi.org/10.1128/mcb.7.4.1393.
Full textAbstract:
Recombinant plasmids of the human c-myc promoter-leader region and the bacterial chloramphenicol acetyltransferase (cat) gene were constructed. After transfection into different rodent and human cells, the 862-base-pair (bp) PvuII fragment carrying both c-myc promoters and 350 bp of the untranslated leader conferred 1/15 to 1/30 of the CAT activity mediated by the simian virus 40 promoter. The presence of additional sequences upstream of the PvuII fragment had an overall negative effect on c-myc promoter activity detectable by titration analysis with small amounts of transfected plasmid DNA. The analysis of numerous deletion constructs in the c-myc promoter-leader region as well as S1 mapping experiments demonstrated that the high CAT activity depended largely on the presence of the second promoter. By cotransfection of c-myc-cat constructs with plasmids carrying different parts of the c-myc promoter locus, targets for positively acting cellular factors were identified. Two positive regulatory elements were mapped within the 862-bp PvuII fragment. One was localized within the 248-bp PvuII-SmaI fragment -101 to -349 bp upstream of the first cap site and the other within the 142-pb XhoI-NaeI fragment of the first exon, comprising positions -95 to +47 relative to the second cap site. We conclude that the dual promotor of the human c-myc gene represents a strong eucaryotic promotor regulated by cooperation of positively and negatively acting cellular transcription factors.
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Warner, J. R., G. Mitra, W. F. Schwindinger, M. Studeny, and H. M. Fried. "Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover." Molecular and Cellular Biology 5, no. 6 (June 1985): 1512–21. http://dx.doi.org/10.1128/mcb.5.6.1512-1521.1985.
Full textAbstract:
The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome. The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids. In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene. Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription. (i) Several ribosomal proteins were synthesized in substantial excess. However, the excess ribosomal protein was rapidly degraded. (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently. We provide evidence that this was due to inefficient initiation of translation. (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA. We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes. Finally, the accumulation of each ribosomal protein was regulated independently. In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.
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Warner, J. R., G. Mitra, W. F. Schwindinger, M. Studeny, and H. M. Fried. "Saccharomyces cerevisiae coordinates accumulation of yeast ribosomal proteins by modulating mRNA splicing, translational initiation, and protein turnover." Molecular and Cellular Biology 5, no. 6 (June 1985): 1512–21. http://dx.doi.org/10.1128/mcb.5.6.1512.
Full textAbstract:
The rate of accumulation of each ribosomal protein is carefully regulated by the yeast cell to provide the equimolar ratio necessary for the assembly of the ribosome. The mechanisms responsible for this regulation have been examined by introducing into the yeast cell extra copies of seven individual ribosomal protein genes carried on autonomously replicating plasmids. In each case studied the plasmid-borne gene was transcribed to the same degree as the genomic gene. Nevertheless, the cell maintained a balanced accumulation of ribosomal proteins, using a variety of methods other than transcription. (i) Several ribosomal proteins were synthesized in substantial excess. However, the excess ribosomal protein was rapidly degraded. (ii) The excess mRNA for two of the ribosomal protein genes was translated inefficiently. We provide evidence that this was due to inefficient initiation of translation. (iii) The transcripts derived from two of the ribosomal protein genes were spliced inefficiently, leading to an accumulation of precursor RNA. We present a model which proposes the autogenous regulation of mRNA splicing as a eucaryotic parallel of the autogenous regulation of mRNA translation in procaryotes. Finally, the accumulation of each ribosomal protein was regulated independently. In no instance did the presence of excess copies of the gene for one ribosomal protein affect the synthesis of another ribosomal protein.
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Gyuris, J., and E. G. Duda. "High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion." Molecular and Cellular Biology 6, no. 9 (September 1986): 3295–97. http://dx.doi.org/10.1128/mcb.6.9.3295-3297.1986.
Full textAbstract:
After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.
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Gyuris, J., and E. G. Duda. "High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion." Molecular and Cellular Biology 6, no. 9 (September 1986): 3295–97. http://dx.doi.org/10.1128/mcb.6.9.3295.
Full textAbstract:
After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.
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Yu, G. L., and E. H. Blackburn. "Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila." Molecular and Cellular Biology 10, no. 5 (May 1990): 2070–80. http://dx.doi.org/10.1128/mcb.10.5.2070-2080.1990.
Full textAbstract:
The macronuclear rRNA genes (rDNA) in the ciliate Tetrahymena thermophila are normally palindromic linear replicons, containing two copies of the replication origin region in inverted orientation. A circular plasmid containing a single Tetrahymena rRNA gene (one half palindrome) joined to a tandem repeat of a 1.9-kilobase (kb) rDNA segment encompassing the rDNA replication origin and known replication control elements was used to transform Tetrahymena macronuclei by microinjection. This plasmid was shown previously to have a replication advantage over the rDNA allele of the recipient cell strain (G.-L. Yu and E. H. Blackburn, Proc. Natl. Acad. Sci. USA 86:8487-8491, 1990). During vegetative cell divisions, the circular and palindromic rDNAs were rapidly replaced by novel, successively longer linear rDNAs that eventually contained up to 30 tandem 1.9-kb repeats, resulting from homologous but unequal crossovers between the 1.9-kb repeats. We present evidence to show that increasing the number of copies of the replication control regions increases the replicative advantage of the rDNA, the first such situation for a cellular nuclear replicon in a eucaryote.
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Yu, G. L., and E. H. Blackburn. "Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tetrahymena thermophila." Molecular and Cellular Biology 10, no. 5 (May 1990): 2070–80. http://dx.doi.org/10.1128/mcb.10.5.2070.
Full textAbstract:
The macronuclear rRNA genes (rDNA) in the ciliate Tetrahymena thermophila are normally palindromic linear replicons, containing two copies of the replication origin region in inverted orientation. A circular plasmid containing a single Tetrahymena rRNA gene (one half palindrome) joined to a tandem repeat of a 1.9-kilobase (kb) rDNA segment encompassing the rDNA replication origin and known replication control elements was used to transform Tetrahymena macronuclei by microinjection. This plasmid was shown previously to have a replication advantage over the rDNA allele of the recipient cell strain (G.-L. Yu and E. H. Blackburn, Proc. Natl. Acad. Sci. USA 86:8487-8491, 1990). During vegetative cell divisions, the circular and palindromic rDNAs were rapidly replaced by novel, successively longer linear rDNAs that eventually contained up to 30 tandem 1.9-kb repeats, resulting from homologous but unequal crossovers between the 1.9-kb repeats. We present evidence to show that increasing the number of copies of the replication control regions increases the replicative advantage of the rDNA, the first such situation for a cellular nuclear replicon in a eucaryote.
28
Peterson, D. O., K. K. Beifuss, and K. L. Morley. "Context-dependent gene expression: cis-acting negative effects of specific procaryotic plasmid sequences on eucaryotic genes." Molecular and Cellular Biology 7, no. 4 (April 1987): 1563–67. http://dx.doi.org/10.1128/mcb.7.4.1563-1567.1987.
Full textAbstract:
A sequence element within pBR322 DNA mediates a cis-acting negative effect on expression from eucaryotic genes in transient expression assays. The negative element overlaps with sequences that inhibit DNA replication, but its effect is observed in the absence of detectable replication of transfected DNA.
29
Peterson, D. O., K. K. Beifuss, and K. L. Morley. "Context-dependent gene expression: cis-acting negative effects of specific procaryotic plasmid sequences on eucaryotic genes." Molecular and Cellular Biology 7, no. 4 (April 1987): 1563–67. http://dx.doi.org/10.1128/mcb.7.4.1563.
Full textAbstract:
A sequence element within pBR322 DNA mediates a cis-acting negative effect on expression from eucaryotic genes in transient expression assays. The negative element overlaps with sequences that inhibit DNA replication, but its effect is observed in the absence of detectable replication of transfected DNA.
30
Danovich, R. M., and N. Frenkel. "Herpes simplex virus induces the replication of foreign DNA." Molecular and Cellular Biology 8, no. 8 (August 1988): 3272–81. http://dx.doi.org/10.1128/mcb.8.8.3272-3281.1988.
Full textAbstract:
Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.
31
Danovich, R. M., and N. Frenkel. "Herpes simplex virus induces the replication of foreign DNA." Molecular and Cellular Biology 8, no. 8 (August 1988): 3272–81. http://dx.doi.org/10.1128/mcb.8.8.3272.
Full textAbstract:
Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.
32
Akusjärvi, G., C. Svensson, and O. Nygård. "A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay." Molecular and Cellular Biology 7, no. 1 (January 1987): 549–51. http://dx.doi.org/10.1128/mcb.7.1.549-551.1987.
Full textAbstract:
The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.
33
Akusjärvi, G., C. Svensson, and O. Nygård. "A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay." Molecular and Cellular Biology 7, no. 1 (January 1987): 549–51. http://dx.doi.org/10.1128/mcb.7.1.549.
Full textAbstract:
The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.
34
Sandlin, Robin C., and Anthony T. Maurelli. "Establishment of Unipolar Localization of IcsA in Shigella flexneri 2a Is Not Dependent on Virulence Plasmid Determinants." Infection and Immunity 67, no. 1 (January 1, 1999): 350–56. http://dx.doi.org/10.1128/iai.67.1.350-356.1999.
Full textAbstract:
ABSTRACT Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-typeS. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigellaand E. coli.
35
Kipling, D., and S. E. Kearsey. "Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA." Molecular and Cellular Biology 10, no. 1 (January 1990): 265–72. http://dx.doi.org/10.1128/mcb.10.1.265-272.1990.
Full textAbstract:
To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.
36
Kipling, D., and S. E. Kearsey. "Reversion of autonomously replicating sequence mutations in Saccharomyces cerevisiae: creation of a eucaryotic replication origin within procaryotic vector DNA." Molecular and Cellular Biology 10, no. 1 (January 1990): 265–72. http://dx.doi.org/10.1128/mcb.10.1.265.
Full textAbstract:
To investigate how a defective replicon might acquire replication competence, we have studied the reversion of autonomously replicating sequence (ARS) mutations. By mutagenesis of a Saccharomyces cerevisiae plasmid lacking a functional origin of replication, we have obtained a series of cis-acting mutations which confer ARS activity on the plasmid. The original plasmid contained an ARS element inactivated by point mutation, but surprisingly only 1 of the 10 independent Ars+ revertants obtained shows a back mutation in this element. In the remainder of the revertants, sequence changes in the M13 vector DNA generate new ARSs. In two cases, a single nucleotide change results in an improved match to the ARS consensus, while six other cases show small duplications of vector sequence creating additional matches to the ARS consensus. These results suggest that changes in replication origin distribution may arise de novo by point mutation rather than by transposition of preexisting origin sequences.
37
Kaufman, R. J., and P. Murtha. "Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells." Molecular and Cellular Biology 7, no. 4 (April 1987): 1568–71. http://dx.doi.org/10.1128/mcb.7.4.1568-1571.1987.
Full textAbstract:
The translational efficiency of mRNA molecules transcribed from plasmid DNA transfected into COS-1 monkey cells can be increased 10- to 20-fold by the coexpression of the adenovirus virus-associated RNAs I and II. Experiments described here demonstrate a similar increase in translational efficiency by the addition of 2-aminopurine, an inhibitor of double-stranded RNA-activated protein kinase, to the culture medium. Both virus-associated RNA and 2-aminopurine presumably exert their effect by alteration of the functional level of eucaryotic initiation factor-2. The translational stimulation mediated by both means is shown to be restricted to the plasmid-derived mRNAs because there is no qualitative or quantitative alteration in host protein synthesis. The results are consistent with models invoking a localized activation of double-stranded RNA-activated kinase leading to a translational block.
38
Kaufman, R. J., and P. Murtha. "Translational control mediated by eucaryotic initiation factor-2 is restricted to specific mRNAs in transfected cells." Molecular and Cellular Biology 7, no. 4 (April 1987): 1568–71. http://dx.doi.org/10.1128/mcb.7.4.1568.
Full textAbstract:
The translational efficiency of mRNA molecules transcribed from plasmid DNA transfected into COS-1 monkey cells can be increased 10- to 20-fold by the coexpression of the adenovirus virus-associated RNAs I and II. Experiments described here demonstrate a similar increase in translational efficiency by the addition of 2-aminopurine, an inhibitor of double-stranded RNA-activated protein kinase, to the culture medium. Both virus-associated RNA and 2-aminopurine presumably exert their effect by alteration of the functional level of eucaryotic initiation factor-2. The translational stimulation mediated by both means is shown to be restricted to the plasmid-derived mRNAs because there is no qualitative or quantitative alteration in host protein synthesis. The results are consistent with models invoking a localized activation of double-stranded RNA-activated kinase leading to a translational block.
39
SCHRELL, Andreas, Juliane ALT-MOERBE, Thomas LANZ, and Joachim SCHROEDER. "Arginase of Agrobacterium Ti plasmid C58. DNA sequence, properties, and comparison with eucaryotic enzymes." European Journal of Biochemistry 184, no. 3 (October 1989): 635–41. http://dx.doi.org/10.1111/j.1432-1033.1989.tb15060.x.
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40
Jackson, Michael W., James B. Day, and Gregory V. Plano. "YscB of Yersinia pestis Functions as a Specific Chaperone for YopN." Journal of Bacteriology 180, no. 18 (September 15, 1998): 4912–21. http://dx.doi.org/10.1128/jb.180.18.4912-4921.1998.
Full textAbstract:
ABSTRACT Following contact with a eucaryotic cell, Yersiniaspecies pathogenic for humans (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) export and translocate a distinct set of virulence proteins (YopE, YopH, YopJ, YopM, and YpkA) from the bacterium into the eucaryotic cell. During in vitro growth at 37°C in the presence of calcium, Yop secretion is blocked; however, in the absence of calcium, Yop secretion is triggered. Yop secretion occurs via a plasmid-encoded type III, or “contact-dependent,” secretion system. The secreted YopN (also known as LcrE), TyeA, and LcrG proteins are necessary to prevent Yop secretion in the presence of calcium and prior to contact with a eucaryotic cell. In this paper we characterize the role of theyscB gene product in the regulation of Yop secretion inY. pestis. A yscB deletion mutant secreted YopM and V antigen both in the presence and in the absence of calcium; however, the export of YopN was specifically reduced in this strain. Complementation with a functional copy of yscB intrans completely restored the wild-type secretion phenotype for YopM, YopN, and V antigen. The YscB amino acid sequence showed significant similarities to those of SycE and SycH, the specific Yop chaperones for YopE and YopH, respectively. Protein cross-linking and immunoprecipitation studies demonstrated a specific interaction between YscB and YopN. In-frame deletions in yopN eliminating the coding region for amino acids 51 to 85 or 6 to 100 prevented the interaction of YopN with YscB. Taken together, these results indicate that YscB functions as a specific chaperone for YopN in Y. pestis.
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Kozak, M. "Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes." Molecular and Cellular Biology 7, no. 10 (October 1987): 3438–45. http://dx.doi.org/10.1128/mcb.7.10.3438-3445.1987.
Full textAbstract:
Simian virus 40-based plasmids that direct the synthesis of preproinsulin during short-term transfection of COS cells have been used to probe the mechanism of reinitiation by eucaryotic ribosomes. Earlier studies from several laboratories had established that the ability of ribosomes to reinitiate translation at an internal AUG codon depends on having a terminator codon in frame with the preceding AUG triplet and upstream from the intended restart site. In the present studies, the position of the upstream terminator codon relative to the preproinsulin restart site has been systematically varied. The efficiency of reinitiation progressively improved as the intercistronic sequence was lengthened. When the upstream "minicistron" terminated 79 nucleotides before the preproinsulin start site, the synthesis of proinsulin was as efficient as if there were no upstream AUG codons. A mechanism is postulated that might account for this result, which is somewhat surprising inasmuch as bacterial ribosomes reinitiate less efficiently as the intercistronic gap is widened.
42
Kozak, M. "Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes." Molecular and Cellular Biology 7, no. 10 (October 1987): 3438–45. http://dx.doi.org/10.1128/mcb.7.10.3438.
Full textAbstract:
Simian virus 40-based plasmids that direct the synthesis of preproinsulin during short-term transfection of COS cells have been used to probe the mechanism of reinitiation by eucaryotic ribosomes. Earlier studies from several laboratories had established that the ability of ribosomes to reinitiate translation at an internal AUG codon depends on having a terminator codon in frame with the preceding AUG triplet and upstream from the intended restart site. In the present studies, the position of the upstream terminator codon relative to the preproinsulin restart site has been systematically varied. The efficiency of reinitiation progressively improved as the intercistronic sequence was lengthened. When the upstream "minicistron" terminated 79 nucleotides before the preproinsulin start site, the synthesis of proinsulin was as efficient as if there were no upstream AUG codons. A mechanism is postulated that might account for this result, which is somewhat surprising inasmuch as bacterial ribosomes reinitiate less efficiently as the intercistronic gap is widened.
43
Axelrod, N. J., G. G. Carmichael, and P. J. Farabaugh. "Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae." Molecular and Cellular Biology 10, no. 3 (March 1990): 947–57. http://dx.doi.org/10.1128/mcb.10.3.947-957.1990.
Full textAbstract:
Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.
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Axelrod, N. J., G. G. Carmichael, and P. J. Farabaugh. "Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae." Molecular and Cellular Biology 10, no. 3 (March 1990): 947–57. http://dx.doi.org/10.1128/mcb.10.3.947.
Full textAbstract:
Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.
45
Musters, W., J. Venema, G. van der Linden, H. van Heerikhuizen, J. Klootwijk, and R. J. Planta. "A system for the analysis of yeast ribosomal DNA mutations." Molecular and Cellular Biology 9, no. 2 (February 1989): 551–59. http://dx.doi.org/10.1128/mcb.9.2.551-559.1989.
Full textAbstract:
To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.
46
Musters, W., J. Venema, G. van der Linden, H. van Heerikhuizen, J. Klootwijk, and R. J. Planta. "A system for the analysis of yeast ribosomal DNA mutations." Molecular and Cellular Biology 9, no. 2 (February 1989): 551–59. http://dx.doi.org/10.1128/mcb.9.2.551.
Full textAbstract:
To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.
47
Kreidberg, J. A., and T. J. Kelly. "Genetic analysis of the human thymidine kinase gene promoter." Molecular and Cellular Biology 6, no. 8 (August 1986): 2903–9. http://dx.doi.org/10.1128/mcb.6.8.2903-2909.1986.
Full textAbstract:
The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.
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Teyssier, Corinne, Hélène Marchandin, and Estelle Jumas-Bilak. "Le génome des alpha-protéobactéries : complexité, réduction, diversité et fluidité." Canadian Journal of Microbiology 50, no. 6 (June 1, 2004): 383–96. http://dx.doi.org/10.1139/w04-033.
Full textAbstract:
The alpha-proteobacteria displayed diverse and often unconventional life-styles. In particular, they keep close relationships with the eucaryotic cell. Their genomic organization is often atypical. Indeed, complex genomes, with two or more chromosomes that could be linear and sometimes associated with plasmids larger than one megabase, have been described. Moreover, polymorphism in genome size and topology as well as in replicon number was observed among very related bacteria, even in a same species. Alpha-proteobacteria provide a good model to study the reductive evolution, the role and origin of multiple chromosomes, and the genomic fluidity. The amount of new data harvested in the last decade should lead us to better understand emergence of bacterial life-styles and to build the conceptual basis to improve the definition of the bacterial species.Key words: alpha-proteobacteria, genome, dynamics, diversity.
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Kreidberg, J. A., and T. J. Kelly. "Genetic analysis of the human thymidine kinase gene promoter." Molecular and Cellular Biology 6, no. 8 (August 1986): 2903–9. http://dx.doi.org/10.1128/mcb.6.8.2903.
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The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.
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Korzh, V., A. Grishin, V. Reshetnikov, and A. Kozlov. "The change of the fate of plasmid pAt153 DNA in fish embryos after insertion of eucaryotic seguences." Cell Differentiation and Development 27 (August 1989): 89. http://dx.doi.org/10.1016/0922-3371(89)90297-9.
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