DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes.

Abstract

Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus Nuclei from various eukaryotic cells injected into frog oocyte nuclei (germinal vesicles) continue to direct synthesis of origin-specific proteins (1). In addition, genes that were not expressed in the donor cell may be activated after injection (2). Purified viral, phage, or plasmid DNAs injected into the nucleus of Xenopus oocytes serve as templates for the transcription of specific RNA sequences (3). The synthesis of 5S RNA (4) and tRNA (5) is greatly stimulated upon injection of the corresponding templates. It has been claimed that, after injection of simian virus 40 (SV40) DNA I (6), much of the virus-specific RNA synthesized in the Xenopus oocyte has the same size and is transcribed from the same region as the late viral mRNAs present in lytically infected monkey cells (7,8), and proteins that migrate on two-dimensional gels in the same manner as the SV40 capsid proteins VP 1 and VP 3 were detected (8).In this article we report that Xenopus oocytes transcribe and translate most or all of the genetic information contained in the SV40 genome. In particular, we observed the synthesis of large (T) and small (t) SV40 tumor antigens having the same characteristics as those synthesized in monkey cells undergoing lytic infection. Injection of polyoma DNA I (6) resulted in the synthesis of three polyoma-specific polypeptides comigrating, during gel electrophoresis, with two minor polypeptides related to polyoma tumor antigen and with the major capsid protein, respectively.MATERIALS AND METHODS SV40 DNA I (covalently closed circular) was isolated from SV40-infected CV-1 African green monkey cells (Flow Laboratories, Irvine, Scotland) 40-48 hr after infection by selective extraction, deproteinized with phenol, and purified by CsCl/ethidium bromide density gradient equilibrium centrifugation (9). Polyoma DNA I was isolated from polyomainfected mouse kidney cell cultures 30-35 hr after infection by the same procedure. For injection, the viral DNA was dissolved in 88 mM NaCl/1 mM KCI/15 mM Tris-HCl, pH 7.6 at concentrations between 300 and 500 Ag/ml. 24 hr in Barth's solution containing 50 units per ml each of streptomycin, penicillin, and kanamycin. Thereafter 1 ml of Barth's solution containing 300 ,uCi of [35S]methionine (500-1000 Ci/mmol, the Radiochemical Centre, Amersham, England) was added and incubation was continued for another 24 hr (1 Ci = 3.7 X 1010 becquerels). The surviving oocytes (60-90%) were washed first with Barth's solution, then with distilled water, and stored at -80'C after removal of the water.For extraction of viral proteins, the oocytes were disrupted in a small Dounce homogenizer in 2 ml of a buffer containing detergent (0.1 M Tris-HCl, pH 9/0.1 M NaCl/5 mM KCI/1 mM CaCl2/0.5 mM MgCl2/0.7 mM Na2HPO4/0.5% Nonidet P-40). The homogenate was sonicated in a MSE ultrasonic disintegrator (Mk 1) for 1 min with an amplitude of 10 Atm and kept on ice for 20 min. After centrifugation at 15,000 X g for 30 min at 40C the s...

Different Potential of Cellular and Viral Activators of Transcription Revealed in Oocytes and Early Embryos ofXenopus laevis

“…Previously, there have been conflicting reports on enhancer activity in Xenopus oocytes. Simian virus 40 (SV40) genes are expressed when SV40 DMA is injected into the oocyte nucleus (de Robertis and Mertz, 1977;Rungger and Türler, 1987). Specifically, the SV40 enhancer in its natural viral context was reported to activate the early promoter (Spinelli and Ciliberto, 1985), while it was inactive in our globin reporter gene constructions (our unpublished data).…”

Regulation of adenovirus transcription by an E1a gene in microinjected Xenopus laevis oocytes.

“…The observation that X. laevis oocytes injected with heterologous molecules are capable of performing the myriad of events from chromatin assembly (27,47) and transcription (7,8,11,15,20,25,26,28,35,40) to protein secretion (24) suggests that they might be useful for studies on the regulation of gene expression. Indeed, they have served as an efficient test system to identify DNA sequences involved in the initiation of transcription (25).…”

“…The fusion was such that the E3 control region is adjacent to the CAT coding sequences and CAT expression is dependent upon transcription initiated from this region. Flanking the 3' end of the CAT sequences are the simian virus 40 Plasmids pJOLC3E and pJN20E are derivatives of pJOLC3 and pJN20, respectively, that contain an additional insert from the SV40 genome which encodes the 72-bp repeat, enhancer sequences (3,19,30). The insert is the HindIlI C fragment from the SV40 genome and was inserted into the unique Hindlll sites of the Ela-containing plasmids.…”

“…Recently, they have been used to examine the mechanisms involved with the transcription of eucaryotic genes. For example, the injection of genes which code for sea urchin histones (15,35), herpes simplex virus thymidine kinase (26), chicken ovalbumin (46), and the papovavirus early and late proteins (11,40) results in the synthesis of not only these specific transcripts but their polypeptide products as well. In the case of the injected papovavirus T antigen and ovalbumin genes, the detection of their encoded polypeptides indicated that oocytes must have correctly processed (spliced) the primary transcripts, although the efficiency with which splicing took place in the injected oocytes was not clear.…”

Cloning of an origin of DNA replication of Xenopus laevis.

“…With these studies in mind we initiated studies on the cloning and characterization of the origins in Xenopus laevts (the African clawed frog), which has been widely used to study the expression (transcription and translation) of foreign genes in oocytes (8)(9)(10)(11)(12). It also has been shown that unfertilized X. laevt eggs have the ability to support DNA replication from injected exogenous DNA (13)(14)(15).…”