Generation of lymphoma-type variant hamster polyomavirus genomes in hamsters susceptible to lymphoma induction.

The hamster polyomavirus (HaPV) induces either hair follicle epitheliomas or lymphomas in either Z3 or HaP respectively. Syrian hamsters. In the lymphomas specifically deleted "lymphoma-type" (lt) HaPV genomes are accumulated. In the present study the temporal pattern of generation of HaPV (lt) DNA was investigated in context of the development of lymphomas in neonatally infected HaP hamsters. The generation of HaPV (lt) DNA was first detectable during the postnatal phase of high level replication of viral DNA in hemopoietic organs (at 7 days post infection), thus clearly preceding the development of overt lymphoma. A variety of HaPV (lt) DNA species is generated in lymphoid cells, but usually only one of them is accumulated to high amounts in lymphoma cells. Furthermore, the pattern of HaPV (lt) and wild-type (wt) DNA was studied in normal and tumor tissues of tumor-bearing hamsters as well as in tumor-free hamsters. In tumor-bearing hamsters predominantly HaPV (lt) DNA species were found in the infected tissues, while HaPV (wt) DNA was detected rarely and only in tumor-free tissues. In contrast, in tissues of tumor-free hamsters HaPV (wt) DNA prevailed over HaPV (lt) DNA species.

In vivo replication of hamster polyomavirus DNA displays lymphotropism in hamsters susceptible to lymphoma induction.

Using the whole body section hybridization technique, we monitored the organ- and age-specific pattern of replication of hamster polyomavirus (HaPV) DNA in a colony of Syrian hamsters, which are susceptible to lymphoma induction. Three phases of viral infection and replication could be distinguished: first, a phase of acute infection characterized by high levels of replication of HaPV DNA in the haemopoietic organs and the liver. This culminated 5 to 7 days post-infection (p.i.); second, at 10 days p.i., a phase of viral clearance became evident; and finally, a third phase reflected both the restriction of HaPV replication in adult hamsters and the accumulation of HaPV DNA at sites of tumour development. A remarkable conformity was observed between the tissue specificity of viral replication and the induced tumour profile: high levels of replication of HaPV DNA were restricted to cells of the haemopoietic system and lymphoid tumours were induced. As shown by in situ hybridization, the viral infection in non-haemopoietic organs was due to the dissemination of HaPV-infected blood cells.

Hamster polyomavirus-encoded proteins: gene cloning, heterologous expression and immunoreactivity.

The hamster polyomavirus (HaPV) is associated with spontaneously appearing skin epithelioma of the Syrian hamster Z3 strain. Virus particles prepared from the skin epithelioma cause lymphoma and leukemia when injected into newborn hamsters from a distinct Syrian hamster colony (HaP); in contrast to the skin epithelioma the hemopoietic tumors are virus free but accumulate viral DNA. To study the humoral immune response of HaPV-infected Z3 hamsters we produced recombinant HaPV proteins in Escherichia coli as beta-galactosidase-, TrpE- and dihydrofolate reductase-fusion proteins or as non-fused proteins. Recombinant plasmids carried segments of all putative early and late HaPV proteins. The recombinant proteins were detected in stained SDS polyacrylamide gels and in Western blots using monoclonal anti-TrpE and anti-beta-galactosidase antibodies and sera of HaPV-infected hamsters. Sera from HaPV-infected Z3 hamsters and crude lysates of all clones were applied to Western blots to characterize the humoral immune response in the animals. HaPV-specific antibodies were found to be directed against early protein segments translated from the first common exon and from the second unique exon of LT and MT, resp., as well as against the late proteins VP1 and VP2/3. The almost complete VP2 was recognized by all sera whereas VP1 was detected only by a half of the sera. Our data suggest the presence of at least 2 immunodominant regions in VP2, one in the C-terminal VP1 and at least 4 in early proteins.

Linkage analysis in German breast cancer families with early onset of the disease, using highly polymorphic markers from the chromosome 17q11-q24 region.

Linkage analysis in German breast cancer families with early onset of the disease by using six markers on chromosome 17q11-q24 has been carried out. In the region between markers D17S250 and GH, three markers showed positive LOD scores at an estimated distance of zero. Evidence for linkage is greatest for D17S250, with a LOD score of 2.42.

Comparison of different in situ hybridization techniques for the detection of human papillomavirus DNA in cervical smears.

Different hybridization methods were used for detection of human papillomavirus (HPV) DNA in cervical smears. The results obtained by filter in situ hybridization (FISH) are consistent with most of the reports recently published. To overcome the unsatisfactory limitations of this method, especially the difficulties to distinguish clearly between positive and negative signals, we developed an in situ hybridization protocol using a cytospin and 35S-labelled as well as biotinylated DNA-probes. For direct comparison of different methods, the samples were obtained from two groups of patients. One group were women with reiterated Papanicolaou smears III, IV; the other were women with reiterated Pap III, IV and additional histological scoring. In all cases but one, the different methods used have shown the same results. In one case the hybridization on slides using 35S-labelled as well as biotinylated probes gave a negative result, whereas the FISH method using a 32P-labelled probe allowed to detect of HPV 16 and 18 DNA only when more than 1 x 10(6) cells were present per filter. Our data demonstrate that in situ hybridization on slides is a specific and sensitive technique, which enables a clear distinction between positive and negative results using a small number of cells and which, especially with biotinylated probes, is suitable for application in routine work.

Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms.

A new promoter located within E6 was mapped in human papillomavirus type 6b (HPV6b)- and HPV11-containing benign genital condylomata (genital warts). The RNA transcribed from this promoter represented the major RNA species colinear with open reading frames E6 and E7 and can encode the E7 protein. No equivalent promoter was active in HPV16-containing cancers and cancer-derived cell lines. In those, the major transcripts contained one of two different introns within E6 and the RNAs could encode two different E6 proteins and E7.

Studies on the SV40-like papovavirus SV40-GBM. III. Propagation at low multiplicities of infection in various human cell lines.

At low multiplicity several human cell lines supported the lytic infection with SV40-GBM better than that with the wild type SV40. The efficiency of viral DNA replication differed in the cell lines used suggesting that specific host cell factors may determine the rate of viral DNA synthesis. Furthermore, the emergence of different DNA defects during propagation of the virus indicates that host cell factors in question might also influence the composition of the viral DNA population.

Studies on the SV40-like papovavirus SV40-GBM. I. Genomic analysis by restriction endonucleases and electron microscopy after propagation in CV-1 monkey cells.

Infection of CV-1 monkey cells with SV40-GBM, a papovavirus isolated from a human glioblastoma multiforme, resulted in the appearance of defective viral DNA molecules. In contrast to SV40 wild-type, two main types of variant DNA molecules could be found after three viral passages at multiplicities of infection of about 10. The molecules of one variant DNA (GBM3-L) were about 19% shorter than the GBM3-H DNA molecules and the DNA of the original GBM isolate, as demonstrated by electron microscopy. Restriction enzyme analysis revealed that GBM3-L DNA had lost both the EcoRI and the HpaII cleavage sites which are located in the late viral genome region. Furthermore, SV40 GBM3-L did not possess the two PvuII sites which are located in the late genome region, and a portion of the GBM3-H and GBM3-L DNA molecules had lost the unique KpnI site. Heteroduplex analysis verified that the rearrangements in the GBM3-L DNA are located only in the late region of this DNA. The possible differences between SV40 wild-type and SV40-GBM are discussed on the basis of these results.

Evolutionary relationships between papovaviruses and their hosts.

The papovaviridae family consists of two genera, the papillomaviruses (PV) and the polyomaviruses (Py-V). Both genera are distinguished by morphological (larger sizes of the PV) and several biological characteristics. The genomes of either of the two genera share highly conserved DNA regions and a common antigenic determinant, located in their major capsid polypeptides. On the basis of these data an evolutionary relationship among the members of PV and Py-V, respectively, has been suggested. No homology has been found for either DNA- or protein sequences between PV and Py-V and the question of a common ancestor for both viral genera remains open. We have started to characterize the genome of a papilloma producing papovavirus of the Syrian hamster (HaPV). Most of the known biological characteristics of the HaPV suggest it should be classified as a papilloma-like virus. However, the molecular weight of about 3.5 X 10(6) daltons found for the circular duplex DNA lies within the range given for SV 40 and polyoma virus (Py). Analysis of the HaPV genome by cleavage with 21 different restriction endonucleases, location of specific binding sites of phage T 4 gene 32 protein and E. coli RNA polymerase on the viral DNA demonstrated that the HaPV differed distinctly from all other currently known papovaviruses. The HaPV genome was also analyzed by filter hybridization and electron microscopy under conditions of varied stringency for nucleotide sequence homology with the genomes of different papovaviruses of both genera. Whereas no homologous DNA regions could be found between the genomes of HaPV and the human PV types 1 and 4, only under nonstringent conditions (Tm-43 degrees C) stable hybrids were formed between HaPV-, SV 40- and the DNA of a PV isolated from Mastomys natalensis (MnPV). On the other hand extensive homology was detected between the genomes of HaPV and Py even under stringent hybridization conditions (Tm-28 degrees C). The homologous DNA segments mapped on the Py and partially on the SV 40 genome were found to be the most strongly conserved DNA regions among the Py-V genus. These results are discussed with respect to a classification of the HaPV within the papovaviridae family.

[Effect of heparin, spermidine and Be2+ ions on the phosphatase and RNAse activity of rat liver cell nuclei]. / Vliianie geparina, spermidina i ionov Be2+ na fosfataznuiu i RNK-aznuiu aktivnost' iader kletok pecheni krys.

Effects of heparin, spermidine, and Be2+ ions on the ATPase and beta-glycerophosphatase and RNA-ase activities of the rat liver cell nuclei were studied. Be2+ was shown to inhibit the ATPase activity and, to a lesser extent, beta-glycerophosphatase activities. Physiological concentrations of heparin and spermidine also lowered the mentioned two activities, as well as the RNAase activity of the nuclei. Evidence is presented for the inhibitory effect of heparin and spermidine on endonucleases.