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H D Robertson

Publications and source records attributed to H D Robertson.

At least 73 records · Page 4Linked to original sources
PubMed1977

A nucleotide sequence from a ribonuclease III processing site in bacteriophage T7 RNA.

Transcription of that portion of the bacteriophage T7 genome encoding early functions yields RNA molecules about 7500 nucleotides long representing this entire early region. These long transcripts can be cleaved in vitro by highly purified Escherichia coli ribonuclease III (endoribonuclease III; EC 3.1.4.24), yielding five messenger RNAs identical to those produced in vivo. During this reaction, a small RNA fragment called F5 RNA is released, which is specified by the region of the T7 genome between genes 1.1 and 1.3. The following sequence of 32P-labeled F5 RNA has been determined using standard RNA sequencing techniques: pU-A-A-G-G-U-C-G-C-U-C-U-C-U-A-G-G-A-G-U-G-G-C-C-U-U-A-G-Uoh. The relative contributions of sequence and structure to ribonuclease III processing signals are considered in light of these findings.

Base Sequence↗
PubMed1977

Interaction of rabbit reticulocyte ribosomes with bacteriophage f1 mRNA and of Escherichia coli ribosomes with rabbit globin mRNA.

We have compared the behavior of a prokaryotic mRNA in a eukaryotic ribosome binding system and of a eukaryotic mRNA in a prokaryotic ribosome binding system. Using (32)P- and (125)I-labeled bacteriophage f1 mRNA, we have shown that rabbit reticulocyte 80S ribosomes can protect specific sequences from pancreatic RNase digestion, including those sequences protected by Escherichia coli ribosomes. We have also found that E. coli ribosomes fail to protect any region of (125)I-labeled globin mRNA. Iodination of the mRNA appeared to have little or no effect on the specificity of binding or protection by the ribosomes of either system.The eukaryotic and prokaryotic systems differ markedly in the ability of the small ribosomal subunits to protect mRNA from nuclease digestion. The regions of phage f1 mRNA protected by E. coli 30S subunits are virtually identical to those protected by the 70S ribosomes. By contrast, rabbit reticulocyte 40S subunits protect substantially larger fragments of mRNA from nuclease digestion than do the 80S ribosomes. These 40S-protected fragments are specific in the case of globin mRNA and overlap the shorter region protected by the 80S ribosomes. However, the 40S-protected fragments of phage f1 mRNA were found to be extremely heterogeneous, reflecting perhaps an important difference between the initial interactions made by these two mRNAs with the ribosomes.

Animals↗
PubMed1976

Cleavage of T4 species I ribonucleic acid by Escherichia coli ribonuclease III.

T4 Species I RNA, a molecule 140 nucleotides in length with some structural features very much like a tRNA, is specifically cleaved by an enzymatic activity in Escherichia coli extracts to give three segments with 19, 48 and 73 nucleotides. We report the purification and characterization of the E. coli RNase which cleaves two 3' phosphodiester bonds of T4 Species I RNA. This reaction has many properties in common with those catalyzed by E. coli RNase III, although the optimal salt conditions for T4 Species I RNA cleavage differ significantly from those for other RNase III-catalyzed reactions. The reaction is not catalyzed by extracts from an E. coli strain lacking RNase III activity. Furthermore, T4 Species I RNA is cleaved by highly purified E. coli RNase III to yield the same three specific fragments. We conclude that this specific cleavage is due to the action of RNase III, and that the requirement for lower ionic strength may reveal further important properties about this RNA processing enzyme.

Cations, Divalent↗
PubMed1976

Potential regulatory roles for RNA in cellular development.

In addition to its widely accepted messenger and structural roles, RNA has been implicated in several other biological events: transfer of specificity of the immune response, interferon induction, and infection of plants by viroids. In none of these cases is the mechanism of RNA action understood. As a result of recent advances in the understanding of RNA metabolism in eukaryotic nuclei and potential specificity of nucleases that cleave such RNA, we have suggested specific ways in which "extra" RNA could be involved in regulation of the development of cells. A survey of literature concerning mammalian cell differentiation both in embryos and in the immune system leads to the observation that there appears to be a line of communication between macromolecules on cell surfaces and the genome. Making the conservative assumption that the DNA retains its integrity throughout development, it seems likely that highly specific signals are sometimes required to transfer information from outside the cell to the DNA,resulting in a change in state of differentiation. We propose a way in which RNA could be utilized in this process.

Animals↗
PubMed1975

Ribonucleic acid processing activity of Escherichia coli ribonuclease III.

We have studied the nuclease activities present in preparations of Escherichia coli RNase III and the "sizing factor" responsible for specific processing of several RNA species. RNase III preparations contain three activities: one which solubilizes stable RNA:RNA duplexes; one which solubilizes the RNA of DNA:RNA hybrids; and one which processes the polycistronic mRNA of bacteriophage T7 in a manner identical with sizing factor. We show that the activity against the RNA of DNA:RNA hybrids can be removed, but that the activity which cleaves RNA:RNA duplexes and that responsible for specific processing of phage T7 polycistronic mRNA appear to be identical by several biochemical criteria. In addition, partially purified enzyme fractions from mutants lacking these two activities contain substantial amounts of activity against the RNA of DNA:RNA hybrids. We have also defined several properties of the two activities solubilize RNA:RNA duplexes and RNA of DNA:RNA hybrids. Average oligonucleotide chain length in an exhaustive digest of double-stranded RNA is about 15 bases, while that in a digest of the RNA in DNA:RNA hybrids is less than 10 bases. Direct analysis shows that both activities cleave RNA chains to yield 5'-phosphate and 3'-hydroxyl termini. All four bases can reside at the 5' end of the resulting oligonucleotides, although both activities show a mild preference for certain bases. These results and previous findings allow us to specify the probably size and structure of potential cleavage sites for these enzymes in biological RNA molecules.

Base Sequence↗
PubMed1975

Sensitive methods for the detection and characterization of double helical ribonucleic acid.

We have evaluated three methods which respond specifically to stable RNA-RNA duplexes and have compared their utility for examining several sorts of nucleic acids. We find that these methods, stepwise chromatography on Whatman CF11-cellulose; digestion with Escherichia coli RNase III; and specific inhibition of globin synthesis in vitro in rabbit reticulocyte lysates, are able to distinguish between stable double-stranded RNA and single-stranded RNA in the expected manner. The most sensitive method, inhibition of globin synthesis, responds to double-stranded RNA concentrations below 0.1 ng per ml. We have used the predominantly single-stranded RNA from several RNA bacteriophages of E. coli to test both the sensitivity and selectivity of these methods. The three viral RNAs tested contain low levels of double-stranded RNA which can be readily removed, leaving RNA which is not recognized as double-stranded RNA, despite indications from physical and sequencing studies that secondary structure is present. In particular, a potential hairpir loop of known sequence has been isolated from phage f2 RNA. Its properties were found to depart significantly from those of RNA-RNA duplexes by those two of our three methods capable of testing RNA of this size. Analysis of two eukaryotic mRNA populations by these methods was complicated by the presence of poly(A). Synthetic poly(A) chromatographs like double-stranded RNA on cellulose CF11 columns, and we could distinguish it from reovirus double-stranded RNA only at elevated temperatures.

Animals↗
PubMed1975

The characteristics of inhibition of protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates.

All types of double-stranded RNA (DSRNA) tested inhibit protein synthesis in rabbit reticulocyte lysates. The inhibition is characterized by its strongly biphasic kinetics, and can be enhanced by preincubation of the lysate with dsRNA in the absence of protein synthesis. Only properly and extensively matched dsRNA (greater than about 50 base pairs) has this property; no form of DNA, single-stranded RNA or even RNA-DNA hybrids act as inhibitors in this way. The cause of the inhibition appears to be a failure of initiator tRNA to associate with native ribosomal subunits in the initiation process (Darnbrough, C., Hunt, T., and Jackson, R. J. (1973) Biochem. Biophys. Res. Commun. 48, 1556-1564). We have shown that this block is not accompanied by stable association of dsRNA with the ribosomes. There are several reasons to believe that the mechanism of action of dsRNA may be complex with the possible involvement of at least one catalytic step. First, the lysate is inhibited by levels of dsRNA at which ribosomes are present in 100-fold excess over base pairs of dsRNA present. Second, high concentrations of dsRNA (greater than 10 mug per ml) are not inhibitory, but can in some, but not all experiments, reverse the inhibition caused by lower levels of dsRNA. Third, a lysate which has been inhibited by dsRNA, when mixed with a fresh lysate will inhibit synthesis in the mixture much more severely than would be expected from the concentration of dsRNA now present. These results indicate that low levels of dsRNA promote the formation of an inhibitor which may exist in two forms: one that is reversible by high levels of dsRNA and one that is irreversible.

Animals↗
PubMed1975

Blood transfusions in elective operations: comparison of whole blood versus packed red cells.

For some years the use of packed red cells in elective operations has been encouraged on an empirical basis. From the clinical and laboratory information gathered in this prospective, randomized, but unblined study of 294 patients subjected to elective general, thoracic, or gynecologic operations, the use of packed cells in such situations appears rational. Such practices minimize demand upon a unique human resource, may give patients fewer undersirable elements in blood transfusions, and do not increase the morbidity or mortality of patients receiving transfusion.

Adult↗