"Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication.

Abstract

Simian virus 40 (SV40) induces tumor (T)antigen formation, chromatin replication, and mitosis in primary mouse kidney cells arrested in Go phase of the mitotic cycle. The temporal and quantitative relation between these early virus-specific reactions led to the hypothesis that the early SV40 mRNA contains information necessary for T-antigen formation and induction of cellular DNA synthesis. To get direct experimental evidence for this hypothesis, the early strand of SV40 DNA was transcribed in vitro by Escherichia coli DNA-dependent RNA polymerase and the SV40-specific cRNA was transferred by microinjection into epitheloid cells of confluent primary mouse kidney cultures. T-antigen formation and stimulation of DNA synthesis were investigated in the recipient cells. The experimental results obtained agree with the hypothesis that T-antigen is a viruscoded protein and that the early virus-specific mRNA contains information necessary for stimulation of cellular DNA replication in the arrested cells.The infectious agent of simian virus 40 (SV40) is the virus DNA. The manner in which the virus genes are expressed is highly cell dependent. Cells of the native host (monkey cells) support early and late viral gene expression at a high efficiency (productive infection) while mouse kidney cells are nonpermissive for SV40 (abortive infection) (1, 2). However, the following sequence of events can be observed in both productive and abortive infected cells: (a) synthesis of early virus-specific mRNA, (b) formation of tumor (T)-antigen, (c) increase of cellular RNA synthesis, (d) induction of chromatin replication and mitosis (3).The temporal and quantitative relation between the synthesis of the early virus-specific RNA and the subsequent sequence of events led to the hypothesis that the early virusspecific RNA may contain information necessary for T-antigen formation and chromatin replication (3,4).In the present study, we have tested this hypothesis by a more direct experimental approach. For (Amersham Buchler,15 Ci/mmol). The nucleoside triphosphates were added after preincubation of SV40 DNA and RNA polymerase at room temperature for 10 min. After 60 min of incubation at 370, 10 ,g of electrophoretically purified DNase (Worthington) was added to the reaction mixture and further incubated for 30 min at 37°. Then 0.1 X SSC and 5% sodium dodecyl sulfate were added to a final concentration of 0.01 X SSC and 1% sodium dodecyl sulfate. The assay was extracted two times with 1 volume of phenol-hydroxyquinoline (80% phenol in H20 vol/vol and-l% hydroxyquinoline weight/volume) (12) and precipitated from the aqueous phase at -20°with 2 volumes of ethanol (95%) and 'Ao volumes of 1 M NaCl. To further reduce traces of any contaminating SV40 DNA, the cRNA preparation was subsequently subjected to Cs2SO4 equilibrium density gradient centrifugation (see Fig. 1) (6, 13). The average yield of cRNA was 80-100 Mug with a specific activity of 1.5 to 2 X 105 cpm/,g of cRNA. Self-an-366

Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes

“…injection of substances into nuclei of living cells, has repeatedly proved to be a valuable assay of diverse nuclear functions, compatible with cell viability and the continuation of nuclear activities (e .g. Graessmann and Graessmann, 1976;Mertz and Gurdon, 1977;DeRobertis and Mertz, 1977;Trendelenburg et aI., 1978;Gurdon and Melton, 1981;SollnerWebb and McKnight, 1982;Etkin, 1982;Renkawitz et aI. , 1982;Green et aI., 1983;Birchmeier et aI., 1984).…”

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

“…Needle microinjection of the affinity-purified anti-cdc25C antibody was carried out as described (Graessmann and Graessmann, 1976).…”

Effect of oncogenic virus on muscle differentiation.

“…If replicating TS-transformed cells after 8 The question whether some events which control differentiation can modulate the course of viral-induced transformation is an important one and the answer to this question may throw some light on the mechanism of transformation as well as differentiation. There are several reports that cells in muscle cultures can be infected with either DNA or RNA viruses (1)(2)(3)(4)(5)(6). However, muscle cultures, and even "muscle clones" consist of a heterogeneous population of cells in different compartments of the myogenic and fibrogenic lineages, and it is by no means clear whether these different phenotypes respond to the viruses in the same manner (7).…”