Plasmids

Publication Title: 
Bioscience, Biotechnology, and Biochemistry

Transfection of nearly senesced human fibroblasts with plasmids encoding HPV16 E6 protein or dominant-negative p53 mutants greatly increased their colony-forming ability. Isolated colonies with these plasmids showed extension of lifespan compared to those with a control plasmid. These data demonstrate that p53 plays a major role in senescence in normal human fibroblasts.

Author(s): 
Ide, A.
Fujii, M.
Nakababashi, K.
Ayusawa, D.
Publication Title: 
Clinical Cancer Research: An Official Journal of the American Association for Cancer Research

PURPOSE: To test ribozymes targeting mouse telomerase RNA (mTER) for suppression of the progression of B16-F10 murine melanoma metastases in vivo. EXPERIMENTAL DESIGN: Hammerhead ribozymes were designed to target mTER. The ribozyme sequences were cloned into a plasmid expression vector containing EBV genomic elements that substantially prolong expression of genes delivered in vivo. The activity of various antitelomerase ribozymes or control constructs was examined after i.v. injection of cationic liposome:DNA complexes containing control or ribozyme constructs.

Author(s): 
Nosrati, Mehdi
Li, Shang
Bagheri, Sepideh
Ginzinger, David
Blackburn, Elizabeth H.
Debs, Robert J.
Kashani-Sabet, Mohammed
Publication Title: 
Cell

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast.

Author(s): 
Szostak, J. W.
Blackburn, E. H.
Publication Title: 
Nucleic Acids Research

We examined structural properties of poly d(C4A2).d(T2G4), the telomeric DNA sequence of the ciliated protozoan Tetrahymena. Under conditions of high negative supercoiling, poly d(C4A2).d(T2G4) inserted in a circular plasmid vector was preferentially sensitive to digestion with S1 nuclease. Only the C4A2 strand was sensitive to first-strand S1 cutting, with a markedly skewed pattern of hypersensitive sites in tracts of either 46 or 7 tandem repeats.

Author(s): 
Budarf, M.
Blackburn, E.
Publication Title: 
Nature

Mutating the CAACCCCAA sequence in the RNA component of telomerase causes the synthesis in vivo of new telomere sequences corresponding to the mutated RNA sequence, demonstrating that the telomerase contains the template for telomere synthesis. These mutations also lead to nuclear and cell division defects, and senescence, establishing an essential role for telomerase in vivo.

Author(s): 
Yu, G. L.
Bradley, J. D.
Attardi, L. D.
Blackburn, E. H.
Publication Title: 
Cell
Author(s): 
Blackburn, E. H.
Publication Title: 
Molecular Biology of the Cell

Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres. We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1. Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants. Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant.

Author(s): 
Lin, Jue
Smith, Dana L.
Blackburn, Elizabeth H.
Publication Title: 
Proceedings of the National Academy of Sciences of the United States of America

A circular plasmid containing a complete Tetrahymena thermophila rRNA gene (rDNA), with a tandem repeat of a 1.9-kilobase-pair segment encompassing the replication origin and the rRNA promoter, and a polylinker in the 3' nontranscribed spacer, was used to transform T. thermophila by microinjection. Most (20/21) stable transformants contained only recombinant linear palindromic rDNA molecules carrying rDNA sequences from both the donor plasmid and the recipient cell, as shown previously.

Author(s): 
Yu, G. L.
Blackburn, E. H.
Publication Title: 
Nature

Mutating the CAACCCCAA sequence in the RNA component of telomerase causes the synthesis in vivo of new telomere sequences corresponding to the mutated RNA sequence, demonstrating that the telomerase contains the template for telomere synthesis. These mutations also lead to nuclear and cell division defects, and senescence, establishing an essential role for telomerase in vivo.

Author(s): 
Yu, G. L.
Bradley, J. D.
Attardi, L. D.
Blackburn, E. H.
Publication Title: 
Proceedings of the National Academy of Sciences of the United States of America

We transformed the ciliate Tetrahymena thermophila by microinjection of circular plasmids containing the ribosomal RNA gene (rDNA). In the somatic macronucleus of Tetrahymena, the rDNA is in the form of linear palindromic molecules. The rDNA molecules from the C3 strain have a replication advantage over rDNA from both B strain and the C3 rDNA mutant rmm1. We constructed two circular plasmids carrying replication origin sequences from C3 rDNA and a point mutation (Pmr) in the 17S rRNA gene that confers resistance to the antibiotic paromomycin.

Author(s): 
Yu, G. L.
Hasson, M.
Blackburn, E. H.

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