Daniel M. Brown

Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans.

We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism.

3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR

3-Nitropyrrole and 5-nitroindole have been assessed as universal bases in primers for dideoxy DNA sequencing and in the polymerase chain reaction (PCR). In contrast to a previous report, we have found that the introduction of more than one 3-nitropyrrole residue at dispersed positions into primers significantly reduced their efficiency in PCR and sequencing reactions. Primers containing 5-nitroindole at multiple dispersed positions were similarly affected; for both bases only a small number of substitutions were tolerated. In PCR experiments neither base, when incorporated into primers in codon third positions, was as effective as hypoxanthine, which was incorporated in six codon third positions in a 20mer oligomer. However, primers containing up to four consecutive 5-nitroindole substitutions performed well in both PCR and sequencing reactions. Consecutive 3-nitropyrrole substitutions were tolerated, but less well in comparable reactions.

The Mutagenic Action of Hydroxylamine

Publisher Summary This chapter discusses the experimental investigation of hydroxylamine mutagenesis. Hydroxylamine has both strong inactivating and strong mutagenic effects on infectious nucleic acids. It is found that in T4 there is a close correlation between mutagenesis, by hydroxylamine, and, by 5-bromodeoxyuridine, it is noted that an opposite conclusion might be drawn from the results of a clean-growth mutagenesis experiment. Studies of the pH dependence of the mutagenic change are of considerable importance in understanding its mechanism. Reaction of poly C, with hydroxylamine, is considerably slower than that of cytidylic acid. In excess reagent, the reaction proceeds, with first-order kinetics, and the analysis of the partly reacted polymer shows that the appearance of the N4-exchange product (III) is considerably delayed. The kinetics both of guanylate incorporation in the presence of adenosine triphosphate (ATP) and of the adenylate incorporation itself as a function of time of hydroxylamine treatment of the poly C, strongly suggest that the residues of II, the mono-adduct, rather than III in the polymer are responsible for the mutagenic error.

MECHANISM OF THE MUTAGENIC ACTION OF HYDROXYLAMINE.

The reaction of aqueous hydroxylamine (pH 6·5) with polycytidylic acid has been studied by comparing at intervals the loss of ultraviolet light absorption (a measure of the addition of the reagent to the 4,5-double bond) and the ratio of cytidylic to N 6 -hydroxycytidylic acid formed after acid hydrolysis. An initial lag in the production of N 6 -hydroxycytidylic acid is taken, with earlier chemical evidence, to indicate that the primary action of the reagent leads to the formation of 4-hydroxylamino-4,5-dihydrocytosine residues. The latter species is considered to be responsible for replication error in hydroxylamine mutagenesis.

Selective reaction of methoxyamine with cytosine bases in tyrosine transfer ribonucleic acid

Abstract The effect of pH on the rate and product ratio has been investigated for the reaction of methoxyamine with cytidine. In the pH range 5.0 to 5.5, approximately 20% of the cytosine bases in mixed Escherichia coli transfer RNA are reactive towards methoxyamine. The cytosine bases in the su + III tyrosine suppressor tRNA which are reactive towards methoxyamine have been characterized as C-16, 19, 35, 51, 83 and 84. Surprisingly, residue C-33, a base at the 5′ terminal of the anticodon loop, is relatively resistant to modification. These results are discussed in terms of transfer RNA conformation.

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

ABSTRACT Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.

The structure of triphosphoinositide from beef brain

Abstract 1. 1. Triphosphoinositide has been isolated from beef brain by a method which depends upon the extraction, purification and dissociation of a protein-inositide complex. Its structure has been shown to be the same as that of the triphosphoinositide isolated by earlier techniques. 2. 2. Mild quantitative deacylation of the triphosphoinositide gave a watersoluble phosphate ester which contained phosphorus, inositol and α-glycol m the ratio 3:1:1. Removal of phosphate monoester groups produced glycerol 1-(inositol 1-phosphate), while removal of the glycerol moiety by a method involving no phosphate migration gave a product identified as l -myoinositol 1,4,5-triphosphate. 3. 3. The triphosphoinositide was thus shown to be 2,3-di- O -acyl glycerol 1-( l myoinositol 1,(4,5)-triphosphate).

Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues

ABSTRACT RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis.

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.

Mutagenic analogues of cytosine: RNA polymerase template and substrate studies

Abstract Using poly (cytidylate, N (4)-hydroxycytidylate) (20:1) as template for the Micrococcus luteus RNA polymerase, study of the substrate requirements for polynucleotide synthesis showed that with GTP poly rG was formed and that added ATP gave rise to poly (G,A) which, in the limit, had a base ratio approaching that of the template. Poly (adenylate- N (4)-hydroxycytidylate) and poly r(A-U) (both alternating) acted very similarly as templates, polynucleotide synthesis now being dependent on added ATP and UTP (giving alternating poly r(A-U)) rather than GTP and UTP. The specific recognition of the analogue as uracil-like when present in a polynucleotide template and the implications of this for hydroxylamine mutagenesis are noted. N (4)-Methyl- N (4)-hydroxycytidylate when present in a copolymer with cytidylate (1 : 14, 1 : 68) and uridylate (1 : 22) acts as a barrier to transcription. The ability of the two analogue substrates, 5,6-dihydrocytidine-5′-triphosphate and N (4)-hydroxycytidine-5′-triphosphate to replace the normal complementary substrates was studied with poly d(A-T) · poly d(A-T), poly (dA) · poly (dT), poly dA and poly d(T-G) · poly d(A-C) as templates in the same polymerase system. Both substrate analogues replaced the normal nucleoside triphosphates UTP or CTP with varying degrees of efficiency depending on the template. Under conditions in which UTP is a substrate, both analogue nucleoside trisphophates behaved identically and were 12 to 13% as efficient as the normal substrate with poly d(A-T), whereas they were 30% as effective with poly d(A-C). Poly d(T-G) was able to use the analogues 65% as effectively as the normal substrate, CTP. The product in each case was the alternating copolynucleotide. Poly dA and poly (dA) · poly (dT) did not direct the incorporation of 5,6-dihydrocytidine-5′-triphosphate. The recognition patterns are discussed in the light of the amino-imino tautomeric states of the pyrimidine bases.