Lanelle G. Gafford

Lability of Host-Cell DNA in Growing Cell Cultures Due to Mycoplasma

In HeLa-cell cultures chronically infected with Mycoplasma, (PPLO) host-cell DNA is unstable as detected by incorporation of H3-or C1 i-thymidine into DNA and subsequent release into the medium as acidsoluble radioactivity. This characteristic can be transmitted to PPLO-free cultures of strain L cells by inoculation with preparations of PPLO from the HeLa cells, although chronically infected cultures of L cells continue to multiply. In addition, virus preparations also may carry PPLO contamination through numerous passages.

The high molecular weight of the fowlpox virus genome

High molecular weight DNA has been extracted from fowlpox virus and purified by chromatography on methylated albumin-kieselguhr columns. The DNA begins to elute at a NaCl concentration of 0.73 m and the elution pattern is consistent with whole molecules. Sedimentation coefficients were corrected to standard conditions and infinite dilution. In an analysis of molecular weights calculated with a number of representative equations using 020,w, three agreed closely, giving values which were comparable with the chemical determination of the mass of DNA per particle and data derived from measurement of contour lengths by electron microscopy. A comparison of these methods indicates that fowlpox DNA has a molecular weight of 200 to 240 × 106 daltons; thus it is the largest viral DNA molecule yet reported.

Growth cycle of fowlpox virus and change in plaque morphology and cytopathology by contaminating mycoplasma

Abstract Studies of the multiplication of fowlpox virus (FP) in chick embryo (CE) monolayers indicate that the eclipse period in this system extends for 24–36 hours and the increase of infectious virus is exponential from 36 to 72 hours with a decline in titer by 120 hours. Most of the virus remains cell-associated throughout this period. The kinetics of the in vitro growth curve in cultures dually infected with FP and Mycoplasma gallisepticum are very similar to pure FP infection, but the in vivo infectivity is markedly decreased. Whereas the pock count of FP alone was approximately 1 log lower than the plaque titer, the pock count of the FP- Mycoplasma mixture was at least 5–6 logs lower. The mechanism of this effect of Mycoplasma on the in vivo infectivity of FP has not been elucidated.

Molecular weight determination of fowlpox virus DNA by electron microscopy

Abstract Fowlpox virus (FP) when gently treated with sodium lauryl sulfate (SLS) alone releases part of the viral genome from the capsid. The DNA associated with the degraded virus was measured from electron micrographs and compared with extracted DNA purified by column chromatography. The contour length of the purified molecules was found to be approximately 100 μ, while the DNA attached to the virus ghosts averaged about 72 μ, thus indicating incomplete release of the molecule in the absence of chemical purification. The size of purified molecules derived from measurement of contour lengths approximates that obtained from analytical ultracentrifugation and chemical determination of the mass of DNA per virion. It is concluded that FP contains a single linear molecule of DNA having a molecular weight of nearly 200 × 10 6 daltons and is the largest viral nucleic acid molecule yet known.

Further studies on high molecular weight fowlpox virus DNA and its hydrodynamic properties

Abstract The intrinsic viscosity of fowlpox virus DNA measured at low shear stress in a rotating cylinder viscometer averages 447 dl/g. Calculation of the molecular weight from intrinsic viscosity data gives a value of approximately 200 × 10 6 daltons and agrees well with the molecular weight previously determined by other methods. Fowlpox DNA shows an anomalous behavior after heat denaturation with as much as 50% of the DNA exhibiting a buoyant density in CsCl similar to that of the native DNA. Denaturation in the presence of 1% formaldehyde virtually eliminates this nativelike material. Sedimentation velocity and buoyant density studies on alkaline-denatured fowlpox DNA give no evidence for covalent cross-links in the molecule. Centrifugation of denatured fowlpox DNA in an alkaline CsCl gradient revealed only one band, indicating that there is no significant difference in the guanine + thymine content of the complementary strands.

Alteration of DNA metabolism in fowlpox-infected chick embryo monolayers

Abstract Secondary chick embryo monolayers infected with fowlpox virus incorporate more thymidine into acid-insoluble material than do control cells for the first 48–72 hr after infection. After this time incorporation in the infected cells falls below control levels. Separation of infected cells into nuclear and cytoplasmic fractions reveals that the major portion of the incorporation occurs in the nuclear fraction. Hybridization of DNA from infected cultures shows that little viral DNA is made before 12 hr post infection, while cellular DNA synthesis proceeds at an essentially normal rate during this time. Viral DNA is synthesized at a rapid rate from 12 to 48 hr while cellular DNA synthesis declines. Although little cellular DNA synthesis can be detected after 60 hr, fowlpox infection does not cause a rapid cessation of cellular DNA synthesis as has been shown for other poxviruses.

Biochemical Changes and Incorporation of P32 in Separated Elements of the Hyperplastic Lesion of Fowlpox

Summary Normal control and fowlpox-infected chick scalp were separated by trypsin into connective tissue and epithelium. By appropriate technics the infected epithelial tissue was separated into surface epithelium and follicles. Comparison was made between normal epithelium and connective tissue and between normal and infected tissues with respect to certain lipids and incorporation of P32 into phospholipid. Distinct differences in phospholipid and total, free, and esterified cholesterol were found between normal epithelium and connective tissue. The above components were increased 2- to 3-fold at some interval of time in both infected epithelium and connective tissue. The specific activities of phospholipid P from all tissues were approximate at 3 days but that of normal epithelium and connective tissue fell approximately 50% at 5 and 7 days. In contrast the incorporation of P32 in infected tissues was 1.5 to 1.7 times that of normal controls.