Treatment Of Poly Research Articles

Light-induced free radical alkylation of polynucleotides and their enzymatic digestion

Ultraviolet light-induced free radical alkylation with 2-propanol or D-ribose, initiated with di-tert-butyl peroxide, of poly (G), poly (U20G), and poly(A) led to the substitution of the appropriate group for the H-8 atom of the purines and addition across the 5,6-double bond of the pyrimidines. The alkylated polynucleotides were subjected to nucleolytic digestion with several nucleases. T1-RNase digestion of poly(G) irradiated with 2-propanol gave a mixture of the modified and non-modified mononucleotides. Similarly, pancreatic RNase digestion of the irradiated poly(U20G) resulted in a mixture of the appropriate mononucleotides. A T2-RNase treatment of poly(A) irradiated with 2-propanol gave the modified Ado-21:3'-P, while T2-RNase digestion of poly(A) irradiated with D-ribose led to the cyclic modified mononucleotides, in addition to the modified mononucleotides.

Peptides isolated from Enterobacter nuclease as potential polyamine binding sites

The Enterobacter nuclease, which cleaves RNA between the 3′-phosphate group of cytidylic acid and the 5′-hydroxyl group of adenylic acid, has been shown to be affected by the polyamines, spermidine, spermine and putrescine. These substances enhance the hydrolytic activity of the enzyme against both poly(C) and yeast RNA. Spermidine and spermine also reverse the inhibition of the enzyme by the ordered polynucleotides, apparently by removing them from the surface of the enzyme. Treatment of poly(G)-bound peptides (obtained from tryptic digests of poly(G)-bound nuclease) with an excess of spermidine resulted in the isolation of spermidine-bound peptides. Purification of these peptides through ion-exchange chromatography resulted in the isolation of three spermidine-bound peptides which consisted of 17 residues (6 amino acids), 19 residues (10 amino acids), and 12 residues (9 amino acids). The binding ratio of spermidine to peptides varied from 1:1 to 3:1.

Tritium labelling of nucleic acids accompanied by conversion of cytosine to uracil.

A new method of incorporation of tritium into nucleic acids with an accompanying conversion of cytosine to uracil is proposed. The method is based on the reaction of nucleic acids with bisulfite in the presence of 3H2O. Under certain conditions poly(C) is quantitatively converted to a radioactive poly(U), whereas similar bisulfite treatment of poly(U) does not result in any tritium incorporation. Specificity of the reaction is confirmed by the results of analysis of modified tRNA and rRNA. Incubation of tRNA with bisulfite and 3H2O does not lead to cleavage of the polynucleotide chain. Similar treatment of the denatured DNA results in tritium incorporation into DNA which is accompanied by a conversion of cytosine to uracil. There is virtually no reaction between native DNA and bisulfite. Only certain cytosone residues in yeast tRNAVal/2a interact with bisulfate providing that reaction is carried out under sufficiently mild conditions.

Abnormal base pairing under the influence of nitrogen mustard

Cyclophosphamide, frequently used in tumor therapy, has proved to be mutagenic [l-4], but so far no clear evidence has been obtained in the host-mediated assay as to whether the substance causes point mutations [5]. Therefore we decided to approach the problem biochemically. Ludlum [6,7] has recently shown that the methylation of cytidine-diphosphate, or poly(C), and to a lesser degree that of the corresponding deoxyderivatives, alters the pairing characteristics of this base in such a way that in the transcription and/or polymerization process not only guanine, but also though to a smaller extent uracil and adenine are incorporated into the resulting polymer. Similar observations regarding the misreading of chemically altered poly(C) have been made by Singer and FraenkelConrat [8,9] and also by Means and Fraenkel-Conrat [IO]. On the basis of the results they obtained with several mutagens, these authors suggest that the substitution of position 3 of cytosine by an alkyl group might well represent a mutagenic event, inasmuch as methylated poly(C) shows appreciable ambiguity, thus leading to abnormal base pairing (with simultaneously diminished capacity for normal pairing with guanine). Since cyclophosphamide as such is not an alkylating agent, we utilized 0, /I’-dichlordiethylamine (nornitrogen mustard) (I) in our experiments. This compound has been postulated to be one of the active substances formed in the body after application of cyclophosphamide [ 111. Therefore, because of the alkylating and cross-linking properties of I [ 121, a strong diminution in the template function of poly(C) after incubation with I is by no means surprising. What could not be foreseen, however, was whether the treatment of poly(C) with such a relatively large molecule (large in comparison with methyl groups) would lead to abnormal base pairing. In fact, our findings have shown a very slight tendency towards the incorporation of LIMP, significantly higher than the “spontaneous” rate of abnormal base pairing with UMP, and increasing linearly with the time of incubation.

Analogue de polynucleotides: XV. Substitution du poly G en C-8 par des sels de diazonium

Treatment of poly G with diazotised p-aminobenzene disulphonic acid gives a polymer in which 30 % of the guanine residues are substituted at C-8. This polynucleotide absorbs in the visible region, possesses a hydrogen-bonded secondary structure with the substituted residues looped out and forms a complex with poly C, in which all residues are hydrogen bonded. Protonation gives rise to a new structure as in the case of poly G·poly C. The same reaction has been applied to poly N 2-dimethylguanylic acid.

The efficiency of induction of mutations by hydroxylamine

Treatment of poly C with methoxyamine leads to formation of 5,6-dihydro-6-methoxyaminocytosine residues in the polymer. These direct incorporation of adenylate residues into the poly G synthesized when the modified poly C is used as a template for RNA polymerase. The process is analogous to that of hydroxylamine-induced errors in replication. Comparison of the uptake of [ 14C]methoxyamine by the poly C with the percentage of adenylate incorporated in the poly G shows that the induction of errors is a highly efficient process: every modified cytosine residue directs the incorporation of one adenylate residue. A probable explanation for this is that the incorporation is mediated by hydrogen-bonding between the adenine and a predominant imino tautomer of the cytosine adduct.

Divalent cation-dependent binding of messenger to ribosomal RNA

The binding of messenger RNA to ribosomal RNA in the presence of magnesium ions was studied using synthetic polynucleotides. Poly U binds strongly to ribosomal RNA in 10−2 M -Mg2+, but not in 10−3 M -Mg2+. When ribosomal RNA is present in molar excess over poly U, several ribosomal RNA molecules can bind to a common poly U molecule. If, however, poly U is in excess, aggregates do not appear. Three to four moles of poly U bind per ribosome equivalent of ribosomal RNA; of these, two or three moles bind to 23 s RNA and 1 or 2 moles to 16 s RNA. Poly C also binds to ribosomal RNA, but binding of poly A to ribosomal RNA was not detected. Formaldehyde was used to examine the role of hydrogen-bonding in the interaction. Formaldehyde-treated ribosomal RNA binds poly C, but will not bind poly U. Treatment of poly C prevents its attachment to ribosomal RNA. It is concluded that base-base hydrogen-bonding binds mRNA to ribosomal RNA. In support of this hypothesis, it is shown that poly U and poly C do not compete for binding sites on ribosomal RNA. Further, a random copolymer of U, G, A and C also binds to poly U and poly C.

The effects of hydroxylamine on polynucleotide templates for RNA polymerase

Treatment of poly C† with hydroxylamine reduces its effectiveness as a template for poly G synthesis in the presence of RNA polymerase. GMP incorporation can be recovered, however, in the presence of ATP, although addition of CTP or UTP has no effect. Nearest neighbour analysis shows that adenylate residues are incorporated into the newly synthesized polymer. On the other hand, treatment of poly A with hydroxylamine has little effect on its properties as a template for poly U synthesis, while treatment of poly U leads to an irreversible inactiva-tion. Moreover, the results strongly implicate 4,5-dihydro-4-hydroxylamino-cytosine residues as the species responsible for hydroxylamine-induced mutagenic transitions.