Use of the bovine leukaemia virus LTR U3 promoter for expressing antisense antiviral RNAs and competitive inhibition of viral infection in cell culture.

Use of viral inducible promoters which can be activated by virus-specific transactivator proteins to drive expression of antisense (as)RNA genes appears to be an attractive approach to inhibit virus infections in vivo. To this end, we have constructed an asRNA gene expressed from the bovine leukaemia virus (BLV) U3 promoter that is complementary to the R-U5 region of the BLV genome. This is the region that is most susceptible to inhibition by asRNA.

Physical mapping and homology studies of egg drop syndrome (EDS-76) adenovirus DNA.

Homologous sequences between EDS-76 adenovirus strain 127 DNA and bovine adenovirus DNA were found by the Southern blotting technique and no homology with CELO virus DNA could be detected. These data suggest a genetic similarity between EDS-76 virus and bovine adenoviruses. The fragments generated form EDS-76 adenovirus DNA by eight restriction endonucleases were physically mapped.

Inhibition of bovine leukaemia virus replication by the antisense RNA in cell line CC81.

A model system has been developed for quantitative evaluation of bovine leukaemia virus (BLV) replication in a permanent cell line CC81. Transfection of the BLV DNA into these cells evoked typical signs of retroviral infection: formation of syncytia, manifestation of reverse transcriptase activity and appearance of characteristic budding retroviral particles. To inhibit BLV replication, a recombinant plasmid pAGR with an antisense RNA gene targeted at the R-U5 region (147th-342th nt) of the viral genome has been engineered.

Comparison of adenoviral hexon polypeptides (monomers) and of native hexons (trimers) by SDS-polyacrylamide gel electrophoresis.

Purified hexons of 27 serotypes of human, simian, bovine and avian adenoviruses were analysed by SDS-PAGE. The apparent molecular weights of hexon polypeptides calculated by comparison with 5 non-hexon and 3 sequenced hexon polypeptide markers ranged from 98 kDa (for bovine adenovirus Ad bos7) to 118 kDa (for simian adenovirus Ad sim13; SV36). A stability of native hexon capsomers (trimers) in SDS at room temperature permitted us to resolve native (trimeric) hexon by SDS-PAGE and to distinguish them from denatured (monomeric) hexon polypeptides by electrophoretic mobilities.

Characterization of a series of monoclonal antibodies specific for bovine adenovirus serotype BAV3 hexon antigen and their use in an ELISA for detection of adenoviral hexons.

After immunization of mice with purified hexon (the main capsid antigen) of bovine adenovirus serotype BAV3 we have obtained a set of 16 individual hybridoma clones producing MAb's against BAV3 hexon. All MAb's were shown to belong to immunoglobulin G class. Specificity of the most avid MAb marked B3Hx-1 was tested on a panel of representative hexon antigens from 16 adenovirus serotypes of human and animal origin using several immunoassays.

Inhibition of adenovirus 5 replication in COS-1 cells by antisense RNAs against the viral E1a region.

To study the effect of antisense E1a RNA (asRNA) on adenovirus development, two types of adenovirus 5 E1a antisense constructs have been engineered. One was complementary to the viral DNA region [nucleotide (nt) positions 500-720] regulated by the metallothionein-I promoter, and the other was complementary to the DNA regions (nt positions 630-1570) under control of the long terminal repeat Moloney mouse leukosis virus promoter. Both asRNA constructs were cloned into a plasmid containing the simian virus 40 origin of replication, the gene controlling geneticin (G418) resistance (G418R), and other regulatory elements.

Adenoviruses as vectors for the transfer of genetic information and for the construction of new type vaccines.

At present many types of corpuscular nondefective, conditional-defective and helper-dependent expressing adenoviral vectors are available which can be used in constructing gene-engineered live or inactivated viral vaccines. In particular, promising results have been obtained with live recombinant human adenoviruses expressing the S antigen of hepatitis B virus, capsid protein of rotaviruses and gB protein of herpes virus. These recombinants are proper candidates for testing as corresponding vaccine strains, a good alternative to well-known recombinant vaccine virus.

Nucleotide sequence of integrated hepatitis B virus DNA and human flanking regions in the genome of the PLC/PRF/5 cell line.

Three fragments of cellular DNA containing integrated hepatitis B virus (HBV) sequences have been cloned from the genomic library of a PLC/PRF/5 cell line. The complete nucleotide sequences of the HBV DNA inserts have been determined, including the S gene and the cellular flanking DNA. Viral sequences were found to be fragmented and rearranged.

Transfer of condensed viral DNA into eukaryotic cells using proteoliposomes.

High-molecular-weight viral DNAs have been packed into proteoliposomes prepared by reverse-phase evaporation followed by phospholipid membrane targeting by influenza virus glycoprotein bound to hydrophobic 'anchors'. DNA has been encapsulated in the form of spermine condensates--toroidal structures sized approx. 0.

Analysis of sequences of simian adenovirus SA7 (C8) DNA in transformed rat cells and hamster tumour cells.

Different methods of molecular hybridization were used to study DNA sequences of the highly oncogenic simian adenovirus SA7 (C8) present in the genomes of two transformed rat cell lines and in cells from three hamster tumours induced by adenovirus SA7. The entire DNA or the left-hand terminal SalI C fragment (19.5% of the genome) were employed.

The site-specific deletion in plasmid pBR322.

The formation of a deletion derivative of plasmid pBR322, designated pBR322 delta 1, was observed during cloning of various eukaryotic DNAs, when the BamHI site of the plasmid vector was used for construction of the recombinant molecules. The restriction analysis of six independently isolated pBR322 delta 1 plasmids allowed establishment of their complete identity. Similar deletion derivatives were also formed as a result of transformation of Escherichia coli cells by the linear form of vector pBR322 produced by BamHI cleavage, but not by SalI or HindIII.

Integration of foreign genome fragments into cells transformed or cotransformed with fragmented adenoviral DNA.

The integration of DNA of highly oncogenic simian adenovirus type 7 (SA7) and non-oncogenic human adenovirus type 6 (Ad6) into the genome of newborn rat kidney cells transformed by fragmented DNA preparations was studied using reassociation kinetics and spot hybridization. Transforming DNA was fragmented with the specific endonuclease SalI (SA7) and BglII (Ad6). In contrast to the cell transformation by intact viral DNA, transformation by fragmented DNA resulted in integration into the cellular genome of not only the lefthand fragment with the oncogene but also of other regions of the viral genome.

The distribution of guanine-cytosine pairs in adenovirus DNAs.

The distribution of guanine-cytosine (GC) pairs in the DNA of the highly oncogenic simian adenovirus type 7 (SA7) and the non-oncogenic human adenovirus type 6 (Ad6) has been studied by thermal denaturation and CsC1 density-gradient centrifugation. The differential of the DNA thermal denaturation curves shows the presence of pronounced peaks which indicates uneven distribution of GC pairs along the DNA chains and the presence of regions with GC content from 30 to 74% in SA7 DNA and from 40 to 68% in Ad6 DNA. The DNA restriction fragments obtained by treatment with EcoRI, BamHI, SalI, BglII and HindIII were subjected to CsC1 density-gradient centrifugation.

Analysis of DNA from human adenovirus type 6 with restriction endonucleases HindIII, BglIII and BamHI.

The genome of the type 6 human adenovirus has three restriction sites for R.BamHI, thirteen for R.HindIII and ten for R.

The secondary structure of bacteriophage DNA in situ. VIII. The reaction of sodium bisulphite with intraphage cytosine as a probe for studying the DNA-protein interaction.

To obtain data on the viral nucleoprotein a study has been made of the reaction of sodium bisulphite with cytosine in the intraphage DNA of the phage Sd. The CHlO4 hydrolysates of the bisulphite-modified phage Sd have demonstrated a decrease of 18% in the cytosine content and the presence of the products with the properties of cytosyl-amino acids (the main amino acid responsible for the DNA-protein interaction involving cytosine is lysine). But when prior to hydrolysis the modified phage was disintegrated under mild conditions in 0.

Construction and properties of hybrid plasmids carrying the E. coli gal operon.

A family of hybrid plasmids carrying the entire gal operon of E. coli and designated pgal was constructed in vitro. In the case of pgal 1 (mol.

Biological activity of the intact and cleaved DNA of the simian adenovirus 7.

Only the deproteinized DNA preparations of the simian adenovirus of the type 7 (SA 7) exhibited transforming and tumorigenic activity. The complex of the SA7 DNA with terminal protein (TP) did not exhibit either transforming or tumorigenic activity in cell cultures. In contrast to the transforming potential the infectious titers of the DNA - TP complex for the monkey kidney cells were 30-50 times higher than those of pure DNA.

Physicochemical properties and restriction maps of simian adenovirus type 38 DNA.

The sedimentation constant of simian virus type 38 (SV-38) DNA was estimated to be 31.6S. The intrinsic viscosity of DNA was on average 86.

Biophysical properties of virions of human adenovirus of the type 6 and its DNA.

A study has been made of human adenovirus type 6 (Ad 6) which belongs to the C subgroup of the nononcogenic adenoviruses. The buoyant density of the Ad 6 virions in CsCl gradient was 1.3545 +/- 0.

EcoRI activity: enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.