Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

The preparation of the thymine peptide nucleicacid (PNA) monomer carrying a 2-nitrophenyl group in position4 is described. This monomer is incorporated into PNAoligomers and reacted with amines to yield PNA oligomerscarrying 5-methylcytosine derivatives. During thedeprotection-modification step two side reactions weredetected: degradation of PNA oligomer from the N-terminal residue and modification of N ⁴-tert-butylbenzoyl cytosine residue. Protection of the N-terminal position and the use of N ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

Aldehyde oxidase cross-reacting material in the Aldox n,cin, mal,and lxd mutants of Drosophila melanogaster

Rocket immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldox ⁿ, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldox ⁿ larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldox ⁿ flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extracts. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L.W.B.     

Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

Summary The preparation of the thymine peptide nucleic acid (PNA) monomer carrying a 2-nitrophenyl group in position 4 is described. This monomer is incorporated into PNA oligomers and reacted with amines to yield PNA oligomers carrying 5-methylcytosine derivatives. During the deprotection-modification step two side reactions were detected: degradation of PNA oligomer from theN-terminal residue and modification ofN ⁴-tert-butylbenzoyl cytosine residue. Protection of theN-terminal position and the use ofN ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

Xanthine dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity

Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin ¹ and cin ⁹ mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L. W. B.     

Postsynthetic Modification of Oligonucleotides with Imidazophenazine Dye and its Effect on Duplex Stability

Carboxyalkyl derivative of the intercalating agent imidazo[4,5-b]phenazine was used for the postsynthetic oligonucleotide modification. Model pentadecathymidylate-imidazophenazine conjugate was prepared from 5′-aminoalkyl-modified (dT)₁₅ by using phosphonium coupling reagent BOP in the presence of 1-hydroxybenzotriazole. Spectral-fluorescent properties of the conjugate were studied. The attachment of the dye was found to increase the thermal stability of (dT)₁₅ duplex with poly(dA) by more than 4°C, probably by the intercalation mechanism.