ISSN:
0006-3525
Keywords:
Chemistry
;
Polymer and Materials Science
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Chemistry and Pharmacology
Notes:
An azo pyrimidine nucleotide has been prepared and enzymatically attached to oligo(A) primers. The nucleotide's azo pyrimidine group has previously been shown to initiate polymerization of methacrylate esters designed to bind marker groups for visualization by microscopy. When attached to RNA molecules complementary to a chromosomal DNA segment, these nucleotides may allow localization of the DNA segment following in situ hybridization of the probe, methacrylate polymerization, and marker attachment. Since mRNA molecules of potential interest as probes bear a 3′-poly(A) tail, the modified nucleotides were added to oligo(A) primers as models. First, N4-ureidocytosine nucleotides were enzymatically added to ApApA, (Ap)9A, or [5′-32P]-(pA)10, using the modified cytidine 5′-diphosphate and “primer-dependent” polynucleotide phosphorylase (M. luteus). In the case of the ApApA-primed reaction, the N4-ureidocytosine nucleotides in the product polynucleotide were converted into azo nucleotides by oxidation with N-bromosuccinimide. The other two primers were employed to study the time course of polynucleotide formation and to verify that primer was indeed being utilized by the enzyme. The suitability of the modified nucleotide for in situ hybridization studies was examined. Poly(N4-ureidocytidylic acid) was prepared from poly(C) and semicarbazide by the bisulfite-catalyzed transamination reaction. It was found that 95% of the N4-ureidocytosine nucleotides in this polynucleotide survive the elevated temperatures typically required for DNA:DNA denaturation and RNA:DNA annealing. When poly(N4-ureidocytidylic acid) was mixed with poly(I) in buffered aqueous salt solutions, no evidence for hybridization was found, so binding of the probe RNA to the denatured chromosomal DNA molecule via the modified nucleotides is not expected. Upon oxidation of poly(N4-ureidocytidylic acid) with N-bromosuccinimide, the azo nucleotides were formed, as judged by the appearance of a characteristic peak at approximately 350 nm in the uv-absorption spectrum of the yellow-orange product, azoRNA. The azo nucleotides in azoRNA exhibited the expected acid lability, which is known to be accompanied by 1-glyceryl methacrylate polymerization in the case of the simple azo pyrimidine. Because 1-glyceryl metharcylate bears substituent glycol groups for attaching heavy atoms or fluorescent markers, it is possible that probe RNA molecules bearing azo nucleotides may be useful for localizing low-multiplicity genes along eukaryotic chromosomes.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bip.1981.360201210
Permalink
