Ferenc Solymosy

Diethyl pyrocarbonate in nucleic acid research.

Publisher Summary Interest in the biological and biochemical effects of diethyl pyrocarbonate, (EtOCO) 2 O, was initiated through the introduction of the compound as a cold sterilizer for beverages. The compound emerged in nucleic acid biochemistry through the demonstration of its nuclease-inhibiting property and through the suggestion of exploiting this property in the extraction of undegraded nucleic acids. Since the experimental verification of this suggestion and the elaboration of a method for the extraction of undegraded nucleic acids, based on its use as a nuclease inhibitor, the reagent was found to be useful in a number of laboratories for securing nuclease-free conditions. This chapter describes that (EtOCO) 2 O reacts with single-stranded nucleic acids and nucleic acid constituents. Such reactions were found to lead to a loss of the biological activity of RNA. This reactivity toward RNA has made some authors conclude that the use of this reagent in nucleic acid extraction might be impracticable. Since the reactions of diethyl pyrocarbonate with nucleophiles are not instantaneous, it is found practical with the purpose of systematizing data, to introduce the concept of “dose” of the electrophile ((EtOCO) 2 O. Fianlly, it concludes that (EtOCO) 2 O is not a unique compound, and not a specific enzyme inhibitor in a strict sense. The study of its interactions with biological materials is, therefore, of interest not only to the immediate, practical use of this compound in nucleic acid research, but it also exemplifies the general mechanisms and problems of concern in the confrontation of cell constituents with acylating agents and other electrophiles.

Uridylate-Rich Small Nuclear RNAs (UsnRNAs), Their Genes and Pseudogenes, and UsnRNPs in Plants: Structure and Function. A Comparative Approach

Abstract Uridylate-rich small nuclear RNAs (UsnRNAs) including the major, or abundant (U1-U6), and the minor, or low abundant (U7-U14), UsnRNAs are a distinct class of evolutionarily highly conserved, metabolically stable molecules that are indispensable functional components (in the form of RNP particles, the UsnRNPs) or nuclear RNP complexes in which splicing of pre-mRNA (U1, U2, U4-U6 RNAs) or processing of pre-rRNA (U3, U8, U13, U14 RNAs) takes place. Their genes are transcribed either by RNA polymerase II (U1-U5 RNAs in metazoa) or by RNA polymerase III (U6 RNA), requiring very specific transcription signals. Most UsnRNPs are assembled in the cytoplasm and then are transported to the nucleus (nucleolus) to exert their function there. Inclusion of plant systems into studies of the structure and function of UsnRNAs, of their genes, and of UsnRNPs (1) substantially contributed to the identification of phylogenetically conserved primary and secondary structural elements in the major UsnRNAs, thus permitt...

Diethyl pyrocarbonate, a new tool in the chemical modification of nucleic acids?

Abstract Diethyl pyrocarbonate was shown to react, under appropriate conditions, with nucleic acids. This was indicated by (a) the incorporation of 14C into rRNA upon treatment with [14C]diethyl pyrocarbonate, (b) changes in the ultraviolet absorption spectra of nucleic acids and their components after exhaustive treatment with diethyl pyrocarbonate, and (c) increased resistance to nucleolytic attack of nucleic acids treated with diethyl pyrocarbonate. The reaction depended on (a) the amount of diethyl pyrocarbonate in the reaction mixture, (b) the length of treatment, and (c) the secondary structure of the nucleic acid. In ribosomal preparations diethyl pyrocarbonate preferentially reacted with the protein moiety of ribosomes.

Sequence homologies between a viroid and a small nuclear RNA (snRNA) species of mammalian origin

Viroids are small, covalently closed circular single-stranded RNA molecules [l] with a length of -250-370 nucleotide residues and a highly basepaired rod-like secondary structure [2-61. They are pathogenic to higher plants and seem to replicate the nuclei of their hosts [7,8]. Despite a firm knowledge of the primary and secondary structure of some of them, and rapidly accumulating information about their mode of replication [9-121, practically nothing is known about the mechanism by which they induce disease symptoms. To support the idea that viroids may interfere with host gene expression, the hypothesis was advanced that the viroid RNA could qualify as a class of small nuclear RNA (snRNA) both in terms of.its locus and metabolic stability [13]. At that time no allusion to a possible similarity in primary structure between these 2 molecular entities was.made. A more precise picture emerged when it was shown [14], by comparing published sequences, that potato spindle. tuber viroid (PSTV) contains a stretch of 23 nucleotides that shows homology with the 5’-end of mammalian Ul RNA, which is a member of the snRNA series (Ul-U6) and believed to play a vital role in the splicing of mRNA (151. Thus, it has been proposed that viroids may interfere with the splicing of host pre-mRNA, mediated by the plant equivalent of U 1 RNA. It was expected [ 141 that all viroids should have in common a sequence homologous with the S-end region of this snRNA species. Indeed, the same homology region was later found [5] in 2 additional viroids, chrysanthemum stunt viroid (CSV) and citrus exocortis viroid (CEV). In this paper we show that: (i) A similar homology region exists in another viroid, avocado sunblotch viroid (ASBV) as well; (ii) Th is p ti 1 ar cu ar viroid contains > 60% of the sequence of U5A RNA from Novikoff hepatoma cells; (iii)Sequence homologies between viroids and mammalian snRNAs are more general than has been anticipated.

Sequence homology between potato spindle tuber viroid and U3B snRNA

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.

Chlamydomonas U2, U4 and U6 snRNAs. An evolutionary conserved putative third interaction between U4 and U6 snRNAs which has a counterpart in the U4atac−U6atac snRNA duplex

The spliceosomal UsnRNAs U2, U4 and U6 from the green alga Chlamydomonas reinhardtii (Cre) were sequenced using a combination of RNA and cDNA sequencing methods and were compared to other sequenced UsnRNAs. The lengths of Cre U6 and Cre U2 RNAs are similar to those of their higher plant equivalents. Cre U4 RNA is shorter (139 nt) than its counterpart from higher plants (150-154 nt), and contains stem IV and loop D which are absent, with the exception of the Tetrahymena U4 RNA, from the U4 RNAs of other unicellular organisms studied to date. Base-pairing interactions between U6 and U4 RNAs and between U6 and U2 RNAs, identical to those described for mammalian and yeast systems, are structurally feasible in the Cre system. In addition, based on comparative analyses of the predicted U4/U6 RNA duplex from various species, an evolutionary conserved third putative U6-U4 interaction was found. Interestingly, it can also be formed with the recently discovered U6atac and U4atac RNAs. This is a strong support in favor of the possible biological significance of this third putative interaction. Based on comparative analysis, an extension of the earlier described U6-U2 interaction patterns is also proposed.

Viroid pathogenicity and pre-rRNA processing: A model amenable to experimental testing

Abstract A model based on sequence data and on results from recent literature suggests that viroids (with the probable exception of avocado sunblotch viroid) exert their pathogenic effect by interfering with the normal way of pre-rRNA processing in plant nucleoli. This interference could consist in a strong binding of viroid RNA to internal transcribed spacer 2 (ITS 2), an intron-like sequence between 5.8 S rRNA and 25 S rRNA that is excised during pre-rRNA processing. The formation of such ITS 2-viroid hybrids would then make ITS 2 inaccessible to U3 small nuclear RNA, a molecular species thought to be involved in a reaction leading to the proper excision of ITS 2.

Role of metabolic activation in the sister chromatid exchange-inducing activity of ethyl carbamate (Urethane) and vinyl carbamate

Ethyl carbamate (EC, urethane) at 10(-2) M concentration induced more sister chromatid exchanges (SCEs) in cultured human peripheral blood lymphocytes in the absence of S9 mix than did 10(-2) M vinyl carbamate (VC), a possible proximate carcinogenic metabolite (Dahl et al., 1978) of EC. VC itself doubled SCE frequency over the control. In the presence of native S9 mix from Aroclor-induced rat liver, the SCE-inducing activity of VC was highly increased whereas that of EC was suppressed. This opposite effect of S9 mix on VC and EC seems to be due to two different factors. Activation of VC by the S9 fraction seems to be due to the presence of mixed-function oxidases in the S9 mix, because neither the native S9 fraction in the absence of co-factors nor the heat-inactivated S9 fraction in the incubation mixture led to the activation of VC. Deactivation of EC by S9 mix, on the other hand, seems to involve the presence of excess protein and/or substances of low molecular weight in the incubation mixture, because this deactivating effect did not change considerably when the S9 fraction was supplied in the absence of co-factors or when it originated from non-induced rat liver. Heat denaturation of the S9 fraction led to an increased deactivating effect on the SCE-inducing ability of EC. This result is in line with the assumption that reactive -SH groups in the S9 protein are at least partly responsible for the deactivation of EC by S9. Heat denaturation of the S9 fraction led to an about 1.5-fold increase in reactive -SH groups.

A novel aspect of the information content of viroids

Viroids were found to exhibit a structural periodicity characterized by repeat units of a length of 11 or 12 (potato spindle tuber viroid group and coconut cadang-cadang viroid), 60 (apple scar skin viroid) and 80 (avocado sunblotch viroid) nucleotide residues, respectively. It is suggested that structural periodicity of viroids is an indication of their protein-binding ability.

Molecular characterization of two tomato U6 RNA pseudogenes generated by RNA-mediated mechanisms

Abstract Two pseudogenes of U6 RNA from a tomato genomic library were isolated and sequenced. Both of them contain a full length, rather than a truncated ‘coding region’ and are likely to have been generated by RNA-mediated mechanisms. This is the first report on the occurrence in plants of pseudogenese combining the above two features.