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Various strains of Escherichia coli were grown to a titer of 5 × 108 cells/ml in peptone media. Following osmotic shocks (peptone → water → 0.4 M sucrose) or lysozyme-EDTA treatment in hypertonic media, the spheroplasts were stored at 4° and then tested for competence with ΦX 174 and M 13 phage DNA and M 12 phage RNA. Lysozyme spheroplasts proved most effective and yielded the following maximum infectivities: for one ΦX 174 DNA molecule, 5 × 10-3 infectious DNA centers; for one M 13 DNA molecule, 10-5 infectious DNA centers; and for one M 12 phage RNA molecule, 2 × 10-5 infectious RNA centers. For many spheroplast preparations competence remained nearly constant for one week. One strain, E. coli 112-12 (λh)F+, retained a constant (though low) level of competence for months when frozen in 10 % (v/v) dimethyl sulfoxide. ΠX 174 DNA saturation experiments indicated that 0.05-1% of the lysozyme spheroplasts were competent. Between 12 and 20 minutes after the addition of a non-saturating level of asp labelled ΦX 174 DNA to such competent cell populations at 37°, 10–20% of the label was adsorbed. Noncompetent cell populations adsorbed no more than 4% of the added label. Prolonging the DNA adsorption time did not give a higher efficiency of DNA infection, because the ceils began losing competence through a temperature-dependent process. More than 90% of the infectious ΦX 174 DNA centers remained sensitive to DNAase during the first 20 minutes in DNA adsorption medium. Plating ΦX 174 DNA and M 12 RNA-infected cells on eosin methylene blue agar during this period prevented the formation of phage, whereas the plaque-forming ability of the mature phage was not inhibited. These observations may an unusual permeability of the competent cells. Inhibitors of phage RNA infection were removed by washing the competent cells. DNAase pretreatment of the spheroplasts increased their competence for infectious M 12 phage RNA. [ABSTRACT FROM AUTHOR]

In SV40-transformed culture cells, viral-specific sequences have been found to be covalently linked to host sequences (Sambrook et al. 1968). The most appealing interpretation to explain the presence of SV40-specific sequences in adult mice following infection at the preimplantation stage would be to assume that the viral DNA was integrated at this early stage of development into the host genome and was thus conserved during further development. However, our results do not exclude an extrachromosomal existence of the SV40 genome, for example, as an independently replicating plasmid or as a lytic infection in a few permissive cells. So far our attempts to demonstrate autonomous SV40 DNA replication in early mouse embryos have been unsuccessful. We plan to investigate whether the SV40-specific information can be genetically transmitted from the infected mice to their offspring; chromosomal integration would be proven if transmission of SV40 DNA occurred in accordance with simple Mendelian expectations. The injection of mouse blastocysts with purified SV40 DNA did not detectably interfere with normal development of the embryos to healthy adult mice, which were still tumor-free at one year of age. This was not due to the trivial possibility that the viral DNA did not successfully infect and was eliminated from the injected embryos, as virus-specific DNA sequences were detected in 40% of the infected year-old animals, or in about 25% of DNA preparations extracted from some of their tissues (Table 1). It is nevertheless possible that the animals may not have been old enough to exhibit tumorigenesis of SV40 origin; to test this possibility, the experiment will have to be repeated for longer survival periods. The absence of any obvious signs of expression of viral genetic functions, i.e., tumor formation, up to one year of age of the host is reminiscent of the "cryptic transformants" described earlier (Smith et al. 1972) which harbor SV40 information but behave essentially like normal untransformed cells. Whether transcription or translation of the virus gene can occur in infected mice is presently an open question. Testing for expression of an integrated viral genome in diverse differentiated tissues may provide a useful model system to study the regulation of differentiation. These matters are currently being investigated.

Explanted mouse blastocysts were microinjected in the blastocoel cavity with simian virus 40 (SV40) viral DNA. After surgical transfer to the uteri of pseudopregnant surrogate mothers, approximately 40% of the blastocysts developed to term and became healthy adults without apparent tumors at 1 year of age. Molecular hybridization tests for the presence of SV40-specific DNA sequences were conducted on DNA extracted from various organs of these animals. Between 0.5 and 13 SV40 genome equivalents per diploid mouse DNA value were found in some organs of approximately 40% of the adult survivors; this represents a substantial augmentation of the amount administered per embryo. The results are consistent with the working hypothesis that the SV40 DNA may have been integrated into the host genome; alternatively, the viral DNA may have replicated as an extrachromosomal entity or by lytic infection in a few permissive cells. Persistence of the viral DNA from preimplantation stages to adult life may thus provide a new tool for experimental investigation of vertical transmission and expression of tumor viruses.

Oligomeric forms of simian virus 40 (SV40) deoxyribonucleic acid (DNA) were isolated from monkey kidney cells infected with two plaque morphology mutants of SV40. Recombinant, large clear-plaque-type SV40 was produced in cells productively infected with oligomeric forms of SV40 DNA.