Bryan E. Roberts

Arrangement of messenger RNAs and protein coding sequences in the major late transcription unit of adenovirus 2.

Abstract The messenger RNAs present in HeLa cells late in adenovirus 2 infection have been characterized utilizing three complementary methods of analysis. In each case the mRNAs were defined functionally, by identification of the polypeptides they encode when translated in a reticulocyte cell-free system. The RNAs were examined by (1) electrophoretic fractionation in agarose gels containing methyl-mercury hydroxide to estimate the sizes of the functional RNAs; (2) selection by annealing to viral DNA fragments immobilized on nitrocellulose to define the genomic origin of their constituent sequences; (3) hybrid-arrested translation using viral DNA fragments to locate their protein coding sequence. The results allow the positioning of both the coding and non-coding regions of all the late mRNAs with respect to the DNA. They demonstrate that the mature mRNAs originating in the major late transcription unit fall into five families. Within each family the mRNAs overlap in a staggered fashion, sharing the same 3′ terminus but differing at their 5′ termini. The data also reveal the existence of two new late proteins of 52,000 and 55,000 Mr encoded between map co-ordinates 30 to 34 and permit the positioning of the late RNAs, according to the polypeptides they encode, as follows: 52, 55K · IIIa: III · pVII · V: pVI · II: 100 K/33 K · pVIII: IV. (The colons signify the ends of families; centre dots the junctions within families; the slash an overlap of coding sequences; and the comma, coding sequences which have not been separated in this analysis. A 55 K protein has Mr = 55,000.) Those mRNAs which are either the only transcripts from a specific region (such as those for polypeptides IX, IVa2 and IV) or are the 3′-terminal transcripts within a family (such as those coding for polypeptides IIIa, V, II and pVIII) migrate as discrete species upon electrophoresis in agarose gels containing methyl-mercury hydroxide. However, the 5′-proximal RNAs within a family (such as those encoding polypeptides 52, 55 K, III, pVII, pVI, 100 K and 33 K) exhibit a broad distribution of molecular weights.

Tobacco mosaic virus RNA directs the synthesis of a coat protein peptide in a cell-free system from wheat☆

Abstract Tobacco mosaic virus (TMV) RNA stimulates amino acid incorporation into protein in cell-free extracts from wheat germ, rye embryo and Escherichia coli . The properties of the wheat germ system are examined and the nature of the viral RNA-induced products studied with the aid of a virus mutant carrying a threonine → methionine replacement in its coat protein. A peptide containing this methionine residue is present in tryptic digests of mutant RNA-directed cell-free products, and is absent from digests of wild type RNA-directed products. The undigested cell-free product contains a very large number of polypeptides with molecular weights from 10,000 to 140,000, but little or no synthesis of correct sized coat protein is observed.

[43] Methods utilizing cell-free protein-synthesizing systems for the identification of recombinant DNA molecules

Herein we outline three methods that, when coupled with cell-free protein synthesis, permit the identification of recombinant DNA molecules encoding specific polypeptides. RNAs enriched by fractionation on methylmercury hydroxide agarose gels are used to prepare sequence-specific probes. Recombinant DNA clones thus identified are further characterized as to their encoded polypeptides by either hybrid selection or hybrid arrest of translation.

[33] R-looping and structural gene identification of recombinant DNA

Publisher Summary This chapter describes the R looping and structural gene identification of recombinant deoxyribonucleic acid (DNA). The advent of recombinant DNA technology has made possible studies on the organization and expression of eukaryotic genes. In high concentrations of formamide, ribonucleic acid (RNA)–DNA duplexes have a higher thermal denaturation temperature than the corresponding DNA–DNA duplexes. When DNA and complementary RNA are incubated under these conditions, R loops are readily formed. For the formation of R loops with recombinant DNA, the procedure consists of incubating sufficient quantities of DNA with limited amounts of messenger RNA (mRNA) such that the rate of the reaction is DNA driven. The rate of R-loop formation is a sensitive function of the incubation temperature. The chapter discusses the R looping of histone plasmids.

The application of a Shapiro reaction — Suzuki coupling sequence to the stereoselective synthesis of E-trisubstituted olefins

Abstract A three-component coupling process is described for the rapid, stereoselective synthesis of a wide variety of E-trisubstituted olefins.

The application of the shapiro reaction to the stereoselective synthesis of E-trisubstituted olefins for cation-olefin cyclization by three component coupling

Abstract A method is described for the rapid and stereoselective assembly of E-trisubstituted olefins by a convergent process from three components

Remarkably complex and unpredictable cyclization and rearrangement reactions of cations derived from unsaturated oxiranes

Abstract Lewis-acid catalyzed reactions of unsaturated epoxides 1 and 2 afford the unprecedented products 3 and 5, respectively. Mechanistic pathways for these reactions are presented (Schemes 2 and 4) along with a more fundamental analysis.