Jan Svoboda

Transmission of (LTR, v-src, LTR) without recombination with a helper virus

We have analyzed transmission of (LTR, v-src, LTR) cryptic structure integrated in the H-19 mammalian tumor cell line. From this cell line different isolates of transforming virus were rescued in heterokaryons produced by fusion with chicken fibroblasts infected by replication-competent avian leukosis virus RAV-1. One of them (F6) was used for the transformation of avian cells in the absence of the helper virus. In four transformed cell lines studied, the (LTR, v-src, LTR) structure was again integrated at a unique position in the cell DNA of each line. This indicated that the (LTR, v-src, LTR) structure is transmitted by the helper virus without recombination. This point has been further supported by the finding that a src-containing species corresponding in size to the nonpolyadenylated src mRNA is present in the RNA isolated from the rescued F6 transforming virus which might serve as template for the synthesis of (LTR, v-src, LTR) structure by the reverse transcriptase provided by RAV-1.

Failure to obtain transfection with XC mitochondrial DNA

Abstract The transfecting activity of DNA obtained from mitochondria of XC virogenic rat cells transformed with the Prague strain of RSV was tested. Both mitochondrial DNA isolated with EthBr, containing circular molecules, and total mitochondrial DNA were inefficient in transfecting sensitive chicken fibroblasts in spite of the fact that doses of mitochondrial DNA up to two orders higher than the minimum efficient dose of total XC DNA were used. These findings support the view that a DNA transcript of the RSV genome is not integrated in mitochondrial DNA.

Stability of C-Banded and C-Bandless microchromosomes in clonal sublines of the RVP3 mouse tumor grown serially in vivo

Karylogic studies were performed on three monocellular clones derived from mouse RVP3 cells, which had been originally transformed with the Prague strain of Rous sarcoma virus. All clones that had originally contained a stable number of microchromosomes, continues to retain them after prolonged passage in vivo. Centromeric heterochromatin was absent in 32% of the microchromosomes as revealed by C-banding technique. The stability of microchromosomes either positive or negative for centromeric heterochromatin is discussed in relation to double-minute chromatin bodies found in early passages of RVP3 tumor cells.

Transmission of B77 Virus Double Mutant LA334 Markers by Transfection

Successful transfection of Brown Leghorn chicken fibroblasts was carried out with DNA isolated from duck cells transformed by the LA334 mutant of avian sarcoma virus B77. Transfection of duck cells was negative. The four viruses recovered after transfection were all temperature-sensitive for transformation. Two were fully temperature-sensitive for replication, as shown by analysis of virus replication, by characterization of virus particles produced at the nonpermissive temperature using density gradient centrifugation, and by electron microscopic examination. The other two viruses were only partially temperature-sensitive for replication. The results suggest that both the src and gag regions of the avian sarcoma virus genome are transferred simultaneously during transfection, probably by a single integral provirus copy.

Karyological analysis of Rous sarcoma virus-producing rat-kangaroo cell lines

Karyological analysis of the PtK 1 cell line and of cell lines derived from it which had been productively infected with Rous sarcoma virus (Schmidt-Ruppin strain) revealed heterogeneity among the various stemlines. The 11-chromosome stemlines of all cell lines were composed of several karyotypic variants. The frequency of chromosomal damage in the Rous sarcoma virus-producing cell lines was not increased above the control level in the noninfected cell line.