Fritz Rottman

Effect of the methylation of the 2′-hydroxyl groups in polyadenylic acid on its structure in weakly acidic and neutral solutions and on its capability to form ordered complexes with polyuridylic acid☆

Abstract The influence of the methylation of the 2′-hydroxyl groups in poly A on its structure in weakly acidic and neutral solutions and on its capability to form ordered complexes with poly U has been investigated by ultraviolet and circular dichroism spectroscopy. A major effect of the methylation of the backbone is to increase the thermal stability of the double-stranded, acid structure of poly A, while no significant effect on the single-stranded conformation was observed. The dissymmetric structures, as reflected in the circular dichroic spectra, were virtually identical for poly A and poly Am § for the tightly packed, double-stranded, acid structures (pH 4.6, 0.15 m -KCl) on one hand and for the single-stranded conformations (pH 7, 0.15 m -KCl, 25 °C) on the other. Circular dichroic spectra with characteristics intermediate between those of the tightly packed, double-stranded and the single-stranded structures of poly A and poly Am are obtained in the pH range from 5 to 7 at moderate to high ionic strength (for poly Am e.g. at pH 5.7, 0.15 m -NaCl, 4 to 44 °C). From the circular dichroic spectra and absorbance melting profiles it is tentatively concluded that poly A and poly Am form less tightly packed, less ordered double-stranded structures under these conditions. Methylation of the 2′-hydroxyl groups in poly A has virtually no effect on the thermal stability of its double-stranded complex with poly U, but considerably weakens the attachment of the third strand of poly U in poly(Am + 2U). It is concluded that the presence of an unsubstituted 2′-hydroxyl group in poly A is not essential for the stabilization of the double-stranded conformations of poly A in acidic solutions, for the partially-ordered single-stranded conformation of poly A at neutrality or for the double-stranded complex of poly A with poly U.

The unique conformational stability of poly 2′-O-ethyladenylic acid

Considerable effort has been made to elucidate the role of 2’-OH groups on the stability and ordered conformation of RNA. Earlier it had been postulated that intramolecular hydrogen bonding between the 2’-OH moiety and nearby groups in the polymer chain contributed to this stability [ 1, 21. However, the evidence derived from a study of the physical characteristics of poly 2’Gmethyladenylic acid (poly Am) indicated that 2’-O-methyl groups did not impair the stability of singleor double-stranded polymers [3]. This implies that hydrogen bonding involving the 2’-0H group is not a major factor in the stabilization of ordered structures of polyribonucleotides. Among various other factors to be considered, the steric environment at the 2’-position may influence the conformation of polynucleotides, perhaps through an altered conformation of the pentose ring. In order to investigate this possible steric effect, a polymer containing the bulkier 2’Gethyl group (poly 2’-O-ethyladenylic acid) was synthesized. The temperature absorbance profile of poly 2’Oethyladenylic acid indicates that it has an even greater tendency for ordered structure than poly rA (polyadenylate) or poly 2’Omethyladenylic acid, thus suggesting that the bulkier group does indeed enhance conformational stability.

Synthesis of bovine growth hormone in primates by using a herpesvirus vector

A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders.

The determination of 2′-O-methylnucleosides in RNA☆

Abstract A rapid and sensitive procedure is described for determining the 2′- O -methylnucleoside methylnucleoside composition of an RNA sample. The RNA is enzymatically hydrolyzed to nucleosides and the 2′- O -methylnucleoside fraction is isolated by DEAE-cellulose (borate) column chromatography. Boric acid is removed as its methyl ester and the 2′- O -methylnucleosides are resolved by liquid chromatography in the presence of ethylene glycol. The sensitivity of this method is sufficient to distinguish RNA samples which differ only 2–3% in 2′- O -methylnucleoside composition.

Nuclear restriction of nucleic acids in the presence of ATP

Summary Analysis of the release of RNA from isolated myeloma nuclei in vitro has shown that, in contrast to other systems, release of RNA was not ATP-dependent. Further, when ATP was added to the reaction mixture DNA was released concomitantly. Analysis of synchronized cells indicated that the release of DNA from nuclei in the presence of ATP could not be attributed solely to fragile mitotic nuclei.

Structural parameters of single and double stranded helical polyribonucleotides

Abstract Precise structural parameters of polyribonucleotides single stranded helices are determined as well as those of double stranded helices of poly 2′-O-methyl A and of poly A at neutral and acid pH. Infrared linear dichroism investigations indicate the similarity of the conformation of the sugar-phosphate backbone of these single and double stranded helices. The angles of the phosphate group for single stranded helix at neutral pH is found to be oriented at 48° for the 02P02 bisector and at about 65° for the 02–03 line to the helix axis. Similar values were found for double stranded poly A helix at acid pH. These structural parameters obtained for the first time on single stranded polynucleotide helices are proposed to be valid for other similar helical chains such as poly A segments of nuclear or messenger RNA and single stranded CCA acceptor end of transfer RNA.

Analysis of mRNA 5′-terminal cap structures and internal N6-methyladenosine by reversed-phase high-performance liquid chromatography

Abstract A simple procedure for determining the complete methylation profile of an mRNA molecule in a single chromatographic separation is described. The mRNA is selectively hydrolyzed to its component nucleosides leaving its cap 0 (m 7 GpppN′) or cap 1 (m 7 GpppN′m) structure intact. The hydrolysis products, which can include cap 0, cap 1, 2′- O -methylnucleosides (N″m) of cap 2 (m 7 GpppN′mpN″m) and internal N 6 -methyladenosine, are separated on an octadecyl reverse-phase column using a mobile phase containing acetonitrile and ammonium formate, a weak ion-pairing reagent. methyl - 3 H-labeled poly(A)-containing mRNA is used to demonstrate the efficacy of the procedure.

Gas chromatographic method for determination of 2′-O-methylation in RNA☆

Abstract A technique is described for the determination of 2′- O -methylation in RNA using gas chromatography to quantitate the methanol released by perchloric acid cleavage of the 2′-methoxy group. The technique can be used with small samples of RNA and does not require radioactive labeling. It is shown that base methylated compounds do not interfere with the assay and that the method is accurate over widely ranging contents of 2′- O -methylnucleotides.

Template activity of 2′-O-methylpolyribonucleotides with Pseudomonas putida DNA-dependent RNA polymerase

Summary Pseudomonas putida RNA polymerase can use the single stranded 2′- O -methylpolyribonucleotides, poly(Um) and poly(Cm), as templates for the synthesis of poly(A) and poly(G), respectively. No template activity was detected with the purine-containing 2′- O -methylated homopolymers, poly(Am) and poly(Im). The poly(Am) strand of either poly(Am)·poly(U) or poly(Am)·poly(Um) was not a template for poly(U) synthesis, and did not prevent the poly(U) or poly(Um) strand of the duplex from serving as a template for poly(A) synthesis. The poly(Um) strand of the duplex poly(Am)·poly(Um) was an effective template for poly(A) synthesis, but the poly(Um) strand of poly(A)·poly(Um) was not.

Influence of increasing 2′-O-methylation on the interferon stimulating capacity of poly(rI) · poly(rC)

Abstract A set of 2′- O -methylated derivatives of poly(rI) · poly(rC) have been prepared and studied for interferon inducing and antiviral activities. Introduction of 2′- O -methyl groups in the polycytidylic strand of this duplex impairs the interferon stimulation capacity more than similar modification of the polyinosinic strand.