Strand-length measurements of normal and 5-iodo-2'-deoxyuridine-treated vaccinia virus deoxyribonucleic acid released by the Kleinschmidt method

Abstract

Purified vaccinia virus, which had been grown on chick-embryo chorioallantoic membranes in the presence or in the absence of 5-iodo-2'-deoxyuridine (IUdR), was suspended in 5 m ammonium acetate and subjected to the one-step Kleinschmidt procedure on surfaces of distilled water or salt solutions. Deoxyribonucleic acid (DNA) molecules were clearly revealed, and in many instances accurate length measurements could be made. The longest continuous molecules from normal virus measured 78, 77, and 65 mu. The most frequent length was approximately 30 mu, which corresponds to only one-third to one-half of the total DNA per virus particle predicted from various chemical analyses. These data provide direct evidence that normal vaccinia DNA may occur as a linear molecule of approximately 150 x 10(6) molecular weight units, but, for reasons still unknown, the majority of these molecules appears to break into segments of equal length during release from the virion. There is no evidence for the presence of cyclic DNA. The DNA molecules are typically double-stranded. DNA from IUdR-treated vaccinia presents a markedly different picture: the molecules are mostly fragmented into small pieces, and rosettes or tangled masses equivalent to even one-quarter the length of normal molecules occur very rarely. The possibility is discussed that at least part of the virus-inhibitory effect of IUdR on vaccinia is due to extensive fragmentation of the DNA molecules into which IUdR has been incorporated in place of thymidine.