Selection for an optimal monovoltine life cycle in an unpredictable environment. Studies on the beetleCatops nigricans Spence (Col., Catopidae).

Catops nigricans reproduces in the autumn. Pre-imaginal development is temperature-dependent and takes place during the winter, followed by aestivation in the early adult stage. This summer diapause is obligatory and temperature independent. It synchronizes the monovoltine life cycle with the annual cycle. In three populations collected near Kiel (54°22' N, 10°6' E), Köln (50°54' N, 7°6' E), and Paris (49°25' N, 2°20' E), pre-imaginal development slowed and the duration of summer diapause decreased with increasing latitude. Synchronization of the critical breeding interval with the appropriate environmental conditions was achieved through temperature- and photoperiod-dependent sensitivity of ovipositing adults, through different thermal thresholds in eggs, larvae, and pupae, and through sensitivity to photoperiod in third-instars larvae.C. nigricans copes with the unpredictability of climatic conditions in different ways. The local populations have evolved a mean diapause length which probably adjusts the life cycle in most years to the optimal date for reproduction. The mean diapause length was 77 days for Kiel, 98 days for Köln and 138 days for Paris at 10°C, short-day (=SD).C. nigricans also spreads the risk by varying diapause length. Amongthe progeny of single females the range of diapause duration covered about 70% of the total range of the whole population. The oviposition rate of females confined to subterranean life was the same as females confined to subterranean life was the same as in those living under the influence of a varying photoperiod.C. nigricans should therefore be able to live both in the litter layer of forests and also in the nests and galleries of small mammals.

Molecular heterogeneity of female genital wart (condylomata acuminata) papilloma viruses.

Florid condylomas from various anogenital sites were examined for the presence of papilloma viruses ( HPVs ) using DNA hybridization techniques. All the condylomas contained HPV DNA, but genetic variability was detected in the form of HPV types, subtypes, and possible recombinants. Morphologically similar lesions contained heterogeneous viral populations. Variation was also detected among the HPV genomes in a single patient, indicating the complex etiology of this disease.

Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells.

Using a newly developed method for microfilament isolation (Matsumura, F., Yamashiro-Matsumura, S. and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644), we have analyzed protein composition of microfilaments in "normal" and transformed rat tissue culture cells. They include REF-52 (an established rat embryo cell line) cells, REF-52 transformed by DNA viruses (SV40 or adenovirus type 5), normal rat kidney cells, and normal rat kidney cells transformed by RNA viruses (Kirsten or Rous sarcoma virus). Microfilaments from normal rat culture cells contain three major tropomyosins (apparent Mr = 40,000, 36,500, and 32,400) and two relatively minor tropomyosins (apparent Mr = 35,000 and 32,000). In transformed cells the levels of one or two of the major tropomyosins (Mr = 40,000 and 36,500) are decreased and the levels of one or both of the minor tropomyosins (Mr = 35,000 and 32,000) are increased. These changes in tropomyosin patterns were also observed in temperature shift experiments with rat-1 cells transformed with a Rous sarcoma virus mutant, temperature-sensitive for transformation. Cell-free translation of whole cell mRNA generated similar tropomyosin patterns on two-dimensional gels, suggesting that changes in the pattern of tropomyosin expression were largely effected at the level of RNA rather than by post-translational modification. Such changes in the tropomyosin composition of microfilaments were consistently found to accompany the various morphological alterations associated with transformation. We suggest that alterations in the pattern of tropomyosin expression are involved in, or cause, rearrangement of stress fibers and that this may be responsible (in part) for morphological transformation.

Laryngeal papillomavirus infection during clinical remission.

Biopsy samples from 20 patients with a history of laryngeal papillomas were analyzed by Southern blot hybridization for the presence of human papillomavirus DNA. All papillomas examined contained papillomavirus DNA sequences. Four samples from uninvolved sites in two patients with active disease and eight samples from patients in remission also contained viral DNA. No human papillomavirus DNA was detected in a third patient seen during remission. These results explain the clinical pattern of frequent recurrences even after long-term remission in laryngeal papillomatosis.

Tumorigenicity of SV40-transformed human and monkey cells in immunodeficient mice.

Human cells transformed in vitro by SV40 to the anchorage-independent state rarely form tumors in nude mice and therefore constitute an important exception to the otherwise tight correlation between anchorage independence and cellular tumorigenicity. In this paper we explore a number of possible explanations for this unusual situation. We find that the phenomenon is not restricted to human cells but includes monkey cells as well. The nontumorigenic phenotype of the primate SV40 transformants is highly stable. We are unable, through selection of ever more anchorage-independent lines, to generate a primate SV40 transformant which will grow as a tumor in even the most immunologically crippled animals. One tumor was obtained from SV80 (an SV40-transformed human cell line) following injection into a mouse deficient in both T and B cell functions. However, cell lines derived from this tumor are not significantly more tumorigenic than the SV80 parent. This low incidence of tumor formation is not due to the fact that the primate cells are transformed by nononcogenic defective viral genomes nor to a nutritional inadequacy of the host animal for the growth of human cells. Although a T cell-independent mechanism may be the major mechanism involved in tumor suppression, it is unlikely that this completely accounts for the general lack of tumor growth by most of these cells. It appears that the interaction of SV40 (a primate virus) with primate cells may be intrinsically less oncogenic than its interaction with rodent cells.

Conserved sequences at the origin of adenovirus DNA replication.

The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A . T-rich region which is partially conserved among these serotypes, and a distal G . C-rich region which is less well conserved. The G . C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis.

Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region.

Recombinant plasmids containing either the entire polyoma viral genome or one or the other of the two HindIII fragments of polyoma virus DNA were constructed and cloned in Escherichia coli X1776, and their DNAs were individually tested for the capacity to transform an established line of rat cells. The recombinant plasmids containing the entire polyoma genome and those containing the HindIII-1 fragment of polyoma DNA (45-1.4 map units) efficiently transform rat cells, whereas the plasmids containing the HindIII-2 fragment (1.4-45.0 map units) do not. The properties of many independent transformed cell lines established by infection with the cloned HindIII-1 fragment were determined. In contrast to the parent cell line, rat cells transformed with the cloned HindIII-1 fragment grow to high saturation densities, form colonies with high efficiency in dilute agar suspension, produce high levels of plasminogen activator, and display a disorganized arrangement of actin cables. By all criteria examined, these cells transformed by fragments are indistinguishable from cells transformed by whole polyoma viral DNA. Cellular DNA prepared from many HindIII-1 fragment-transformed cell lines was analyzed for the presence and arrangement of polyoma viral sequences by Southern blot-hybridization. In all cases examined, only those viral sequences contained within the HindIII-1 fragment of polyoma DNA were detected. These data establish a strong correlation between polyoma DNA sequences mapping within a restricted portion of the early region and the induction and maintenance of the transformed phenotype.

Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells.

The ability of the two early simian virus 40 (SV40) coded proteins, the large and small T-antigens, to abortively induce the disappearance of cytoplasmic actin-containing networks in cultured cells has been studied in rat embryo fibroblasts after microinjection of intact SV40 DNA, DNA fragments from the early region of SV40, and a purified SV40 large T-antigen related protein (the D2 hybrid protein) isolated from cells infected with the adenovirus-SV40 hybrid virus Ad2+D2. Injection of either the 107,000-dalton D2 hybrid protein or SV40 DNA from the deletion mutant dl 884 SV40, which lacks part of the region (0.54 to 0.59) encoding small t-antigen, failed to cause any detectable change in the structure of actin cables in recipient cells over a period of 72 h. By contrast, injection of wild-type SV40 DNA or a DNA fragment containing the entire region coding for a small-t antigen leads to the disruption of actin cable networks within 24 h of injection. It appears likely that the SV40 small-t protein is necessary for the abortive loss of actin cables in injected cells. Epidermal growth factor also causes loss of actin cables in rat embryo fibroblasts or Rat 1 cells (an established rat embryo line), but only after exposure of the cells to epidermal growth factor in the culture medium and not after injection of epidermal growth factor into the cells.

Variable defectiveness for lytic growth of the dl 54/59 mutants of simian virus 40.

When viral growth in TC-7 cells is compared with that in the simian virus 40 (SV40) transformed CV-1 line C6 some mutants of SV40 deleted between 0.54 and 0.59 on the standard map (dl 54/59 mutants) give relative bursts similar to those of wild-type strain 776, whereas others grow markedly poorer in the untransformed cell. In general, viruses which are defective by this criterion have been found to produce neither a fragmentary small-t protein nor a mature small-t mRNA.

Retransformation of a simian virus 40 revertant cell line, which is resistant to viral and DNA infections, by microinjection of viral DNA.

We have isolated morphological transformants of cultured cells as dense foci on a monolayer of normal cells appproximately 4 weeks after microinjection of purified simian virus 40 DNA (200 to 400 molecules per cell) directly into the nucleus. Both Rat 1 (an established contact-inhibited rat embryo fibroblast line) and F1' 1--4 (a 5-fluorodeoxyuridine-selected flat revertant from the simian virus 40-transformed 14B cell line) were transformed with an efficiency of 5 to 10% of the cells injected. F1' 1--4 is not susceptible to retransformation by either viral or DNA infection (by calcium phosphate-facilitated cellular uptake), and as a result it had previously been thought to possess a host mutation preventing expression of the simian virus 40 genome.

Transformation of rat cells by the hybrid virus Ad2(2+) HEY.

A set of four isogenic rat cell lines transformed by Ad22+ HEY have been studied. While all of the cell lines synthesize SV40 T antigen, only one expresses adenovirus 2 T antigen: none expresses SV40 V antigen or adenovirus 2 fibre antigen. Three cell lines contain 1 to 2 virus equivalents of SV40 and adenoviral sequences per diploid quantity of rate cell DNA and the fourth line contains five copies of SV40 and 20 copies of the adenovirus genome. At least three of the cell lines contain DNA sequences from the helper adenovirus 2 in addition to sequences from the Ad2+ HEY genome. The patterns of integrated virus sequences are complex suggesting multiple insertions of both adenovirus and SV40 DNA sequences. SV40 can be rescued from three cell lines by fusion with permissive cells.

Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells.

Mutants of SV40 with deletions of various sizes mapping between 0.54 and 0.59 on the genome grow at a rate equal to or slightly slower than that of wild-type virus, in a range of host cells. Their ability, however, to induce transformation in several mouse, rat and rabbit cell lines is impaired. The extent of transformation observed is dependent upon the assay used to measure it, but in general, the ability of the mutants to transform falls as the size of the deletion increases. In addition, rat embryo fibroblasts transformed by deletion mutants have fewer of the characteristics of a fully transformed phenotype (for example, growth in low serum, increased saturation density, growth in semi-solid medium) than those transformed by wild-type virus. During lytic infection, immunoprecipitable T antigen produced by the deletion mutants is of the same size as that seen during infection with wild-type virus, and is present at a similar level. Mutant virus-coded small t protein, however, is reduced in size compared with that from wild-type virus. For each mutant, the reduction in protein size is dependent upon the amount of DNA deleted, but not on the relative position of the deletion in the genome. These results demonstrate that the DNA sequences mapping between 0.54 and 0.59 on the viral genome code for the small t protein, and that SV40-induced transformation is at least partially dependent upon the expression of this protein.