Showing papers by "Phillip A. Sharp published in 1982"
TL;DR: It is concluded that plasmid DNA integrated into Chinese hamster ovary DNA is extremely fluid, as demonstrated by its frequent deletion, and transfected DNA sequences most frequently deleted are also those most frequently amplified on methotrexate selection.
523 citations
TL;DR: High resolution analysis of runoff transcripts showed that the reconstituted system initiated transcription precisely at the adenovirus major late and early region IV promoters.
251 citations
TL;DR: The rat genome contains two segments closely related to a rat α-tubulin mRNA that are a processed α- Tubulin pseudogene and a dispersed repetitive element inserted within it that possibly reflect a common RNA-mediated process of insertion.
Abstract: The rat genome contains two segments closely related to a rat alpha-tubulin mRNA Both have been cloned and complete nucleotide sequences are presented Analysis of the structure and sequence of one of these establishes it as a functional alpha-tubulin gene The second segment is a processed alpha-tubulin pseudogene Comparison of this pseudogene to the mRNA and gene coding for alpha-tubulin strongly suggests that a mature mRNA was involved in its origin Features of the pseudogene and a dispersed repetitive element inserted within it possibly reflect a common RNA-mediated process of insertion
194 citations
TL;DR: Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DH FR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenyation signal.
Abstract: Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.
193 citations
TL;DR: The assembly of hexon, the major capsid protein of adenovirus, was investigated with the use of conformation-specific monoclonal antibodies to reveal the unique nature of this structure, which is a trimer of three identical monomers folded into a highly conserved and stable structure.
101 citations
TL;DR: The generation of mammalian cell lines carrying functional amber suppressor genes is described, which are stable and exhibit normal growth rates.
90 citations
TL;DR: Interestingly, the amplification of an amber suppressor gene in CV-1 cells does not interfere with SV40 production, suggesting that suppression of amber codons may not be very deleterious to mammalian cell metabolism.
Abstract: We describe the synthesis, cloning, expression, and in vivo function of a suppressor tRNA gene in mammalian cells. By using "primer-directed mutagenesis" on a Xenopus laevis tyrosine tRNA gene cloned into the recombinant single-strand phage M13mp5, we have generated an amber suppressor tRNA gene that has a nucleotide change--GTA leads to CTA--in the anticodon sequence. The suppressor (Su) tRNA gene was introduced into monkey kidney cells (CV-1) by using simian virus 40 (SV40) DNA as vector (SV40-tRNATyrSu+). CV-1 cells infected with virus containing the mutant, but not the wild-type, tRNA gene produce a functional amber suppressor tRNA as indicated by suppression of amber mutations in co-infecting adenovirus serotype 2-SV40 hybrids. Further evidence that suppression of these amber mutations is tRNA mediated was derived by isolation of total tRNA from CV-1 cells infected with the SV40-tRNATyr (Su+) recombinant and its use in demonstration of read through of an amber codon during in vitro translation of tobacco mosaic virus RNA in reticulocyte extracts. Interestingly, the amplification of an amber suppressor gene in CV-1 cells does not interfere with SV40 production, suggesting that suppression of amber codons may not be very deleterious to mammalian cell metabolism.
56 citations
TL;DR: The results suggest that the 3′ ends of different precursor RNAs derived from a primary transcript may be formed by different mechanisms, including cleavage of larger precursors and the creation of abundant messenger RNAs for the virion protein hexon and the 100K M r protein.
48 citations
Patent•
23 Mar 1982TL;DR: In this article, a cell line characterized by its production of monoclonal antibodies, demonstrating specific reactivity to an antigenic determinant possessed by a plurality of types of adenoviruses, and a method of isolating such cell lines, and the use of such antibodies for diagnostic and therapeutic purposes as well as for identifying chemical compounds with similar properties, are disclosed.
Abstract: Monoclonal antibodies, and a cell line characterized by its production of such monoclonal antibodies, demonstrating specific reactivity to an antigenic determinant possessed by a plurality of types of adenoviruses, a method of isolating such cell lines, and the use of such antibodies for diagnostic and therapeutic purposes as well as for identifying chemical compounds with similar properties, are disclosed.
33 citations