The E5 gene of bovine papillomavirus type 1 is sufficient for complete oncogenic transformation of mouse fibroblasts.

The transforming 69% fragment of the bovine papillomavirus type-1 genome was inserted into a retrovirus vector, which also expresses G418 resistance, and the resulting construct was used for transfection of psi 2 cells. C127 cells infected with virus-containing medium from G418 resistant psi 2 clones were selected for G418 resistance and/or transformation. G418 resistant cells contained invariably a 4.4 kb provirus. The transformed cells, in contrast, contained either a 2.8 kb or a 5.4 kb provirus. Cells containing the 2.8 or the 5.4 kb proviruses were fully transformed according to several criteria: they had a transformed morphology, formed colonies in soft agar, contained disarranged F-actin cables and induced tumors when injected subcutaneously into nude mice. A molecular analysis of the 2.8 kb provirus showed that it contained only one complete BPV-1 gene, namely E5. Cells transformed by the 2.8 kb provirus contained colinear RNAs as well as subgenomic mRNAs, composed of a retrovirus leader sequence connected to an exon starting at nucleotide 3605 in the BPV-1 sequence. The only papillomavirus protein that can be expressed from these mRNAs is the E5 protein. The results suggest that the 44 amino acid long membrane protein which is encoded by the E5 gene of BPV-1, confers a fully transformed phenotype to immortalized mouse cells in the absence of other viral gene products or papillomavirus regulatory elements.

Bovine papillomavirus oncoprotein E5 affects the arachidonic acid metabolism in cells.

The bovine papillomavirus type 1 (BPV-1) oncoprotein encoded by the E5 ORF is a small highly hydrophobic protein, which is capable of inducing oncogenic transformation of cells. We studied the effect of the BPV-1 E5 protein expression on the arachidonic acid metabolism in monkey (COS1) and human (C33A) cells. At relatively low protein concentrations the phospholipase A(2) (PLA(2)) activity and the arachidonic acid (AA) metabolism are activated. E5 mutant proteins, lacking cysteines responsible for the dimerisation of the protein (C37S, C37SC39S), and truncated E5, lacking the C-terminal region, are non-transforming and unable to stimulate the PLA(2) activity and AA metabolism. The transformation-defective mutant D33V, which does not activate the platelet-derived growth factor receptor (PDGFR), activates AA metabolism like wt E5. Our data suggest that the BPV-1 E5 protein could stimulate the AA metabolism independently of PDGF receptor.

Replication of the bovine papillomavirus type 1 genome; antisense transcripts prevent episomal replication.

A subgenomic fragment, representing 69% of the bovine papillomavirus type 1 (BPV-1) genome, has the capacity to transform mouse C127 cells in vitro and to replicate episomally in these cells. In the present study we have cloned this BPV-1 fragment between two retrovirus-derived long terminal repeats (LTRs) in the two possible orientations. The constructs were designated pMR and pML. The pMR construct contained the BPV-1 genome in the same transcriptional orientation as that of the LTRs whereas the pML construct contained the BPV-1 fragment in the opposite orientation. Both types of construct were capable of transforming mouse C127 cells with approximately the same efficiency. Analysis of the intracellular DNA from cells transformed with pMR and pML revealed that both cell types contained a large number of viral DNA copies. However, whereas the pMR-transformed cell clones contained episomally replicating BPV-1 DNA, the pML-transformed cell clones all contained integrated viral genomes. Analysis of RNA from the transformed cells revealed that the pML-transformed cells, unlike the pMR-transformed cells, contained large amounts of antisense BPV-1 transcripts which presumably interfere with the mRNAs that are required for episomal BPV-1 replication. The results are of importance for the future design of BPV-1 vector constructs.

5 S RNA-protein complex is involved in ribosomal subunit association.

The immobilized tRNA-50 S ribosomal subunit protein (TP50) complex binds the smaller ribosomal subunit. We constructed tRNA . TP50 . 5 S [32P] RNA and tRNA . TP50 . t [32P] RNA complexes and investigated the accessibility of the 32P-labelled tRNAs to ribonuclease T1. It was found that in this complex both 5 S RNA and tRNA are attacked by T1 RNase. In sharp contrast, the addition of 30 S subunit protects 5 S RNA as well as tRNA from degradation. We suggest that 5 S RNA-TP50 complex is exposed to the ribosomal interface and is involved in subunit interaction.

The interaction of ribosomal protein L16 and its fragments with tRNA.

Two large proteolytic fragments of Escherichia coli 50 S ribosomal subunit protein L16 were generated by limited hydrolysis with chymotrypsin (missing 9 N-terminal amino acids) and trypsin (missing 16 N-terminal amino acids). It was found that while intact L16 and its chymotryptic fragment both interact with tRNA (Kd = 5.4 x 10(-7) M), the tryptic fragment does not. These results are interpreted in terms of possible significance of the residues 10-16 in the peptidyl transferase activity.

Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags.

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged, resin-bound DNA-binding proteins. The BPV-1 E2-derived tags can be recommended as useful tools for detection and purification of proteins.

Inhibition of the bovine papillomavirus E2 protein activity by peptide nucleic acid.

The bovine papillomavirus type-1 E2 protein is the master regulator of the papillomavirus transcription and replication, the activity of which is regulated through sequence-specific DNA binding. Peptide nucleic acid (PNA) is a nucleic acid analogue, which associates with high affinity to complementary DNA, RNA or PNA, yielding in formation of stable complexes. The potential use of PNA as a sequence-specific inhibitor of the E2 protein activity is studied in this report. We demonstrate that replacement of one or both DNA strands with the complementary PNA reduced drastically the affinity of the BPV-1 E2 protein to its target site in the direct as well as in competitive binding as shown by in vitro gel-shift assays. We demonstrate that PNA could specifically bind to the double stranded E2 binding site by forming the complex with DNA oligonucleotide. In addition, PNA was able to bind specifically to the E2 binding site within the supercoiled plasmid DNA. Such binding of PNA to the E2 binding site within the origin of replication specifically abolished the activity of the E2 protein in the initiation of DNA replication in vivo.

Replication of a chimeric origin containing elements from Epstein-Barr virus ori P and bovine papillomavirus minimal origin.

The bovine papillomavirus E2 protein is a multifunctional protein that activates viral transcription, co-operates in initiation of viral DNA replication, and is required for long-term episomal maintenance of viral genomes. The EBNA1 protein of Epstein-Barr virus is required for synthesis and maintenance of Epstein-Barr virus genomes. Both viral proteins act through direct interactions with their respective DNA sequences in their origins of replication. The chimeric protein E2:EBNA1, which consists of an transactivation domain of E2 and DNA binding domain of EBNA1 supported the replication of the chimeric origin that contained EBNA1 binding sites in place of the E2 binding sites principally as full-length E2 did in the case of papillomavirus minimal origin. This indicates that the chimeric protein E2:EBNA1 is competent to assemble a replication complex similar to the E2 protein. These data confirm the earlier observations that the only part of E2 specifically required for its activity in replication is the N-terminal activation domain and the function of the DNA binding domain of E2 in the initiation of replication is to tether the transactivation domain of E2 to the origin of replication.

Roles of the hinge region and the DNA binding domain of the bovine papillomavirus type 1 E2 protein in initiation of DNA replication.

The bovine papillomavirus (BPV-1) E2 protein is the regulator of extrachromosomal replication of papillomaviruses. The mutants with C-terminal truncations and in-frame internal deletions were constructed to study the role of structural domains of E2 in the initiation of DNA replication. We show that the replication initiation function of E2 is absolutely dependent on the ability of the protein to bind to DNA. Our study also confirms the borders of the functional domains of the E2 protein; residues 1-192 form the activation domain and residues 310-410 the DNA binding-dimerization domain. Some critical length and flexibility, but not the particular amino acid sequence between these two functional domains is required for the activity of the protein to support replication. The hinge region, including the major phosphorylation sites of E2, is also dispensable for the mediation of attachment of the BPV1 genome to the mitotic chromosomes.

Hepatitis C virus genotypes in Estonia.

Distribution of hepatitis C virus (HCV) geno(sub)types among 215 Estonian patients hospitalized with acute or chronic hepatitis and with HCV RNA-positive sera was investigated. For genotyping, both multiplex PCR with subtype-specific primers of the core region and RFLP analysis of cDNA of the 5' NCR region were used. These two methods permitted a correct characterization of genotypes, a more truthful characterization of mixed infections, and combined use of single-tube performances. They revealed, respectively, 200 and 202 (93.0% and 93.9%) HCV-positive samples of sera, subtype 1a- 0.9% and 0.9%, 1b- 56.3% and 64.2%, 3a- 13.9% and 22.3%, 2a- 6.5% and 5.6%, type 4 0.5% and 0%, mixed infections- 13.5% and 0%, and unidentified- 1.4% and 0.9%. In the majority of cases (84.7%) both methods gave completely or partially concordant results; in mixed infections, as determined by subtype-specific PCR, only one subtype was revealed by the RFLP method. In the remaining 15.3% of the cases (Ohno- 7.0%, RFLP- 8.3%) only one of the methods was positive. The epidemiological analysis of the dynamics of the subtypes' relative participation may indicate increasing 3a and decreasing 1b subtype infection during recent years.

Diversity of Helicobacter pylori genotypes among Estonian and Russian patients with perforated peptic ulcer, living in Southern Estonia.

To compare the genomic variation of Helicobacter pylori in samples obtained from patients with perforated peptic ulcer, living in the same area of Estonia but belonging to different nationalities, 50 non-consecutive patients (32 Estonians and 18 Russians) admitted in the Tartu University Hospital in 1997-1999 were studied. Gastric samples of antral mucosa were obtained during operation and analysed histologically and with PCR for detection of different genotypes of H. pylori (cagA and vacA s and m subtypes). Among the 50 perforated peptic ulcer patients with histologically proven H. pylori colonisation no sample of gastric mucosa showed the s1b subtype of the vacA gene. The perforated peptic ulcer patients were mainly infected with cagA (82%) and s1 (98%) genotypes of H. pylori. The distribution of s1a/m1, s1a/m2 and s2/m2 subtypes of vacA genes was statistically different in Estonian and Russian patients (P<0.05). In conclusion differences in the distribution of vacA s and m subtypes of H. pylori were revealed between Estonian and Russian patients with perforated peptic ulcer from Southern Estonia.

Opioid antagonist naloxone potentiates anxiogenic-like action of cholecystokinin agonists in elevated plus-maze.

This study investigated the interplay of cholecystokinin (CCK) and endogenous opioid peptides in the regulation of anxiety. The acute administration of non-selective CCK agonist caerulein (1 and 5 microg/kg) and a selective CCK(B) receptor agonist BOC-CCK-4 (1, 10 and 50 microg/kg) induced a dose-dependent anxiogenic-like action in the plus-maze model of anxiety. BOC-CCK-4 displayed a similar efficacy with caerulein, indicating that the described effect was mediated via CCK(B) receptor subtype. The opioid antagonist naloxone itself (0.5 mg/kg) did not change the exploratory activity of rats in the plus-maze. However, the combination of naloxone with the sub-effective doses of caerulein (1 microg/kg) and BOC-CCK-4 (1 microg/kg) induced a significant inhibition of exploratory behaviour in rats. Accordingly, CCK and endogenous opioid peptides have an antagonistic role in the exploratory model of anxiety in rats.

[Expression of human tissue plasminogen activator by recombinant cell lines from various animals]. / Ekspressiia tkanevogo aktivatora plazminogena cheloveka rekombinantnymi liniiami kletok razlichnykh zhivotnykh.

A number of recombinant cell lines which produce human tissue plasminogen activator (tPA) was obtained using different hosts--mouse, rat, hamster, simian and human cell lines. All types of recombinant lines secreted active r-tPA into conditioned medium. A slight difference between molecular weights of secreted variants of r-tPA was mediated by the different mechanisms of protein modification. Treatment of some recombinant cell lines with different substances resulted in increased levels of r-tPA production.

[Recombinant murine cell lines, transformed by various vectors based on bovine type 1 papillomavirus and expressing human tissue plasminogen activator. II. Analysis of cell lines, producing recombinant tissue plasminogen activator]. / Rekombinantnye linii kletok myshi, transformirovannye raslichnymi vektorami na osnove bych'ego papillomavirusa tipa 1 i ékspressiruiushchie tkanevoi aktivator plazminogena cheloveka. II. Analiz kletochnykh linii, produtsiruiushchikh rekombinantnyi tkanevoi aktivator plazminogena.

We have characterized a number of recombinant cell lines established with BPV1 and Lx1 (containing duplication of LCR-E6-E7 sequence) vectors on the basis of C127 cells. It had been shown that Lx1 based vectors possess the higher number of intracellular copies than analogous vectors on the basis of wtBPV, and most part of them is integrated into the host genome. Using various concentrations of heavy metal salts we have developed the optimized procedure for induction of recombinant tPA synthesis which is controlled by the mouse MT1 promoter. A 8-fold increase of rtPA concentration was reached in the course of induction. It had been shown that native and non-glucosylated forms of recombinant and human tPA are identical in their properties.

[Binding of Escherichia coli 50S ribosomal subunit proteins with two large 5S RNA fragments]. / Sviazyvanie belkov 50S subchastitsy ribosomy Escherichia coli s dvumia krupnymi fragmentami 5S RNK.

Two fragments containing sequences from 1-41 nucleotide (small fragment) and from 42-120 nucleotide (large fragment) were isolated from E. coli 5S RNA T1 RNase partial digest. Affinity chromatography of 50S ribosomal proteins on the immobilized 5S RNA fragments revealed the ability of the large fragment to give a complex only with protein L25. The small fragment did not bind ribosomal proteins. The intact and reassociated 5S RNA forms a complex consisting of proteins L5, L18, L25.

[Protein L16 of the Escherichia coli ribosome: possible role in protein biosynthesis]. / Belok L16 ribosomy Escherichia coli: vozmozhnaia rol' v biosinteze belka.

We show that Escherichia coli 50S ribosomal subunits depleted of protein L16 can nevertheless catalyze the transfer of the peptide moiety from fMet-tRNA to puromycin, being, however, unable to use a fragment CACCA-Phe as an acceptor substrate. On the other hand, we found that protein L16 as well as its large fragment (amino acids 10-136) both interact with tRNA in solution (Kd approximately 10(-7) M). Moreover, L16 interacts with CACCA-Phe in solution as well as protects 3' end of tRNA from the enzymatic degradation. We suggest that L16, although not being the peptidyl transferase as such, is involved in the binding of the 3' end cytidines of tRNA into the ribosomal A site.