Chromosomal locations of satellite DNA in the MSWBS ascites tumor cell line.

In situ hybridization of mouse satellite complementary RNA to the chromosomes of the mouse ascites tumor line MSWBS revealed that the distribution of satellite DNA sequences paralleled the location of constitutive heterochromatin as defined by the C-banding technique.

Alimentary fibropapilloma in cattle: a spontaneous tumor, nonpermissive for papillomavirus replication.

Fibropapillomatosis of the upper alimentary canal of cattle is described. The tumors, found in the esophagus, esophageal groove, and rumen, showed involvement of the subepithelial fibroblasts as well as of the squamous epithelial layer. Although the fibropapilloma cells harbored multiple episomal copies of the genome of bovine papillomavirus type 2 (BPV-2) easily detected by hybridization techniques, no mature virus could be isolated from these lesions or seen by electron microscopy, and no viral antigen could be detected by immunohistochemical methods. It would appear, therefore, that within the limitations of the techniques employed the alimentary canal epithelium and the underlying fibroblasts, while allowing BPV-2 DNA replication, are nonpermissive for the expression of the viral vegetative functions and that transformation of the epithelial cells, like transformation of fibroblasts, can take place in the absence of infectious viral progeny.

Detection of Epstein-Barr virus (EBV) DNA sequences using in situ hybridization.

In situ hybridization was used to detect Epstein-Barr virus (EBV) DNA sequences under conditions where the virus DNA is replicating spontaneously and where it is induced to do so following superinfection. The in situ reaction itself is influenced by several parameters, analogous to conventional nucleic acid hybridization, consideration of which should help to optimize the designing of in situ hybridization reactions in general. Both EBV complementary RNA (cRNA) and EBV DNA synthesized in vitro can efficiently detect the virus DNA sequences in situ. The findings presented here can therefore be utilized in both the study of EBV-cell interactions and, more generally, in studies using in situ hybridization as a general approach.

Detection of virus and cellular-determined antigens in situ using [125I]protein A and autoradiography.

The present paper describes the use of [125I]Protein A, isolated from Staphylococcus aureus, in detecting antigen-antibody complexes by autoradiography on single cells. the method is relatively quick, reproducible, potentially more sensitive than immunofluorescence, and should be useful in combination with conventional radioimmuno-assays. We have used it to detect the cellular expression of IgM, kappa, lambda, and beta 2-microglobulin, as well as the expression of Epstein-Barr Virus (EBV)-associated antigens expressed in human lymphoblastoid cell lines.

Abortive expression of the Epstein-Barr virus (EBV) cycle in a variety of EBV DNA-containing cell lines, as reflected by nucleic acid hybridization in situ.

A variety of Epstein-Barr virus (EBV) DNA-containing cell lines have been tested for the expression of the EBV-associated antigens EBNA (nuclear antigen), EA (early antigen), and VCA (viral capsid antigen), and for the presence of cells containing disproportionate amounts of EBV DNA. The antigen tests utilized immunofluorescence and 125I-labelled antibodies combined with autoradiography. EBV-DNA was detected by in situ hybridization with 3H-labelled EBV RNA complementary to P3HR-1 EBV DNA (P-EBVcRNA). The P-EBVcRNA has been shown to represent the majority of the P3HR-1 EBV DNA sequences. It was concluded that EBV DNA-containing cell lines can be divided into those that express only EBNA, those that express EBNA and EA and those that express EBNA, EA and VCA and also contain cells that undergo disproportionate EBV DNA synthesis. Consequently, in some cell lines there is an abortive expression of the EBV cycle in that some cells spontaneously express EA but fail to continue further to viral DNA synthesis. A similar pattern can be found after experimental induction of the EBV cycle, suggesting that related mechanisms govern the spontaneous expression of the EBV cycle and the extent of its inducibility.

Effect of mitomycin C and hydroxyurea on the expression of the Epstein-Barr virus cycle following P3HR-1 superinfection.

Epstein-Barr virus (EBV) DNA synthesis following EBV P3HR-1 superinfection of the Burkitt lymphoma cell line, Daudi, is refractory to mitomycin C and hydroxyurea at concentrations which inhibit cell DNA synthesis to greater than 98% of control values. Further, neither mitomycin C nor hydroxyurea inhibit virus antigen synthesis following P3HR-1 superinfection.

Detection of virus-specific DNA and RNA base-sequences in individual cells transformed or infected by adenovirus type 2.

By means of 3H thymidine-labelled adenovirus 2 DNA, adenovirus-specific DNA sequences have been localized in individual nuclei of adenovirus 2- or 12-infected cells, and adenovirus-specific RNA sequences have been detected in the permissive cells as well as in cells transformed by adenovirus type 2. The ability to detect such specific virus RNA base-sequences in particular, by in situ hybridization, should be useful in studying the transcriptional specificities of tumours and neoplasms.

A cutaneous fibropapilloma from a red deer (Cervus elaphus) associated with a papillomavirus.

A cutaneous fibropapilloma was found on a Scottish red deer (Cervus elaphus), and a papillomavirus was isolated from it. The virus appeared to be related to bovine papillomavirus type 1 (BPV1) or type 2 (BPV2) because: (i) it cross-reacted in peroxidase-antiperoxidase tests with antisera raised against these virions; (ii) BPV1 and BPV2 DNAs cross-hybridized to the red deer papillomavirus in situ; and (iii) BPV1 and/or BPV2 DNA cross-hybridized to the red deer papillomavirus DNA on Southern blots under conditions of high stringency. These tests also revealed a unique restriction enzyme cleavage pattern for the red deer papillomavirus DNA.

Induction of Epstein-Barr virus early antigen, virus capsid antigen and virus DNA synthesis by mitomycin C.

The effect of mitomycin C was tested on Epstein-Barr virus (EBV) antigen and virus DNA synthesis in producer and non-producer cell lines. We found that producer, but not non-producer cell lines, are inducible for early antigen and virus capsid antigen synthesis. Although host DNA synthesis is effectively inhibited by over 99% in the presence of mitomycin C, virus DNA synthesis in the producer lines is unaffected and induced in parallel with the virus antigens.

Detection localization of Epstein-Barr virus-associated early antigens in single cells by autoradiography using 125I-labeled antibodies. (With 1 color plate).

A characteristic feature of virus-transformed cells is the expression of virus-specific antigens usually detectable by immunological or radioimmune procedures. We report here an autoradiographic method for the detection and localization of such antigens in individual cells which combines the cellular specificity and radiolabeling sensitivity of the above procedures. As a test system we have studied the reaction between virus antigen in some Epstein-Barr virus (EBV) DNA-containing cell lines and anti-virus antibody specificities in certain human sera. EBV DNA-containing cell lines express an EBV-determined nuclear antigen (EBNA) and some lines also express the EBV-associated antigens, early antigen (EA) and virus capsid antigen (VCA), in a minority of cells in the population. By employing appropriate 125I-IgG-labeled human sera, we clearly show that EA can be detected without difficulty in cell lines known to spontaneously express this antigen. Moreover, the specificity and sensitivity of the present method is such that low levels of EA cand be detected at cellular concentrations which remain undetectable by conventional immunofluorescence.

Influence of temperature on the detectibility and chromosomal distribution of specific DNA sequences by in situ hybridisation.

Hybridising certain AT-rich satellite complementary RNAs (cRNAs) to their homologous chromosomal DNA sequences at different temperatures of incubation results in a different dispersion of autoradiographic label throughout the karyotypes. The temperature at which most label, or cRNA-DNA hybrid formation, exists corresponds to the optimal rate temperature for the hybridisation of these same satellite cRNA-DNA hybrids as determined by RNA excess filter hybridisation. It is likely that the in situ hybridisation results can therefore be explained by the fact that there is a similar temperature-dependence on the rate of hybrid formation for both in situ and RNA excess hybridisation. This should have important implications for the designing of in situ hybridisation experiments in general.

Autoradiographic detection of Epstein-Barr virus (EBV)-associated early antigen in a variety of EBV DNA-containing lymphoblastoid cell lines previously designated as nonproducers.

By using autoradiography combined with 125I-labeled IgG prepared from human sera containing high anti-EA (early antigen) antibody titers, EA has been detected in a low fraction of cells in a variety of EBV DNA-containing cell lines previously designated as negative for this antigen. These positive cell lines are highly inducible for EA following treatment with iododeoxyuridine (IUDR) and also contain multiple copies of the virus genome on average. The correlation between IUDR inducibility and spontaneous expression of EA, in particular, suggests that the ability to express EA in both situations is interrelated.

Viral DNA sequences detected in a hamster liposarcoma induced by bovine papillomavirus type 4.

Following intradermal inoculation of bovine papillomavirus type 4 (BPV-4) into a Syrian hamster, a liposarcoma developed at the inoculation site 20 months later. The DNA of this tumour contained multiple copies of the BPV-4 genome which existed in a free unintegrated state. Unintegrated viral DNA and viral DNA isolated from virus particles from bovine alimentary tract papillomas revealed identical cleavage patterns with CpG methylation-resistant and -sensitive restriction enzymes: apparently there was no gross methylation of CpG sites in either case. The entire BPV-4 genome appeared to be represented in the tumour DNA.

Molecular hybridization of RNA and DNA in situ" visualization at the electron microscope level.

Electron microscopic visualization of molecular hybrids formed in situ is feasible at the present time. It can be accomplished by two alternative approaches. In one, the in situ hybridization is carried out on ultrathin sections of target embedded in glycol methacrylate. In the other, whole cells are used for hybridization and they are subsequently prepared for electron microscopy. The choice of the method to be adopted depends on the type of target tissue. When there is a choice, the second approach seems preferable. Some of the important technical steps in the hybridization procedure, such as DNA denaturation in ultrathin sections, have been discussed and attention has been drawn to practical problems that may arise during the preparatory steps. Our light microscope experiments demonstrate that preparations made after glutaraldehyde fixation have a lower hybridization efficiency than those fixed with 3 : 1 methanol-acetic acid. Attempts are therefore being made to explore the possibility of using methanol-acetic acid for electron microscope in situ hybridization. First results of straight-forward fixation show that the preservation of nuclear structure may be fairly satisfactory for the purpose. However, the cumultative effects of subsequent treatments in the procedure still remain to be examined. For electron microscope autoradiograph (EM ARG) of hybridized preparations, the most suitable emulsion at present appears to be Ilford L4. Various factors conductive to optimum resolution consistent with maximum efficiency in this emulsion have been pointed out. Practical problems that may arise in autoradiographs of hybridized preparations such as background and variation of grain density in adjacent sections have also been considered.

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.

Unintegrated viral DNA sequences in a hamster tumor induced by bovine papilloma virus.

A fibrosarcoma was induced in a hamster by bovine papilloma virus type 2 (BPV2). The content of BPV2 DNA sequences was measured by DNA-DNA and cRNA-DNA hybridizations. The tumor contained approximately 300 BPV2 genome equivalents per cell. Southern blot hybridization indicated that the viral DNA was in free form, the entire genome most likely being present. In situ hybridization with BPV2 cRNA showed that multiple genome copies were present in each cell. Neither virus particles nor virus coat antigens could be detected in the tumor. A cell line was established from the fibrosarcoma, and the cells contained multiple copies of the BPV2 genome. The latter was in free form, and all of the DNA sequences appeared to be present in multiple copies and in all cells. An extensive search failed to reveal the presence of virus or viral antigens.

The presence of bovine papillomavirus type 4 DNA is not required for the progression to, or the maintenance of, the malignant state in cancers of the alimentary canal in cattle.

In the Western Highlands of Scotland there is a very high incidence of alimentary cancers in cattle. The carcinomas of the upper alimentary canal are found in association with virus-induced benign papillomas, and transformation of papillomas to carcinomas has been observed. Strong circumstantial evidence suggests that the progression to malignancy is due to the interplay between the virus, bovine papillomavirus type 4 (BPV-4), and carcinogen(s) present in bracken fern, which infests the marginal upland grazing grounds. The carcinomas are often accompanied by adenomas and adenocarcinomas of the lower bowels. To elucidate the role of the virus in the transformation process, we have analysed several malignancies of the alimentary canal, and have detected the viral genome in only one case of transforming papilloma of the oesophagus and one case of carcinoma of the tongue. We conclude that, although required for the induction of papillomas, the presence of the BPV-4 DNA is not necessary for the progression to, or the maintenance of, the transformed state.