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Biomedical subjects

J Ríman

Publications and source records attributed to J Ríman.

At least 37 records · Page 2Linked to original sources
PubMed1988

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. II. Search for common sites of proviral integration in tumour DNA.

To demonstrate the existence of a common site of integration in independent tumour clones, restriction mapping of the vicinity of integrated MAV-2 proviruses in nephroblastoma DNA was performed, using Southern hybridization with an MAV-2 specific probe U3(pAT). The results have shown that (1) nephroblastomas are of semiclonal origin. (2) Nephroblastoma cells contain an average of 5 clonally located integrated proviruses per diploid genome; they do not contain any detectable amount of non-integrated proviruses. (3) In the DNA from independent nephroblastoma clones, there appear at an increased frequency Tth111I fragments of 14.6 and 17.8 kb that hybridize with the U3(pAT) probe. Considering a random selection of integration sites, such coincidence is of little probability. Thus we suppose that these fragments represent a common site(s) of integration with an MAV-2 proviral insert. Two hypotheses concerning possible mechanisms of nephroblastoma induction are discussed: proto-oncogene insertional activation and anti-oncogene insertional inactivation.

Animals↗
PubMed1987

Monoclonal antibodies against RNA-dependent DNA polymerase from myeloblastosis-associated viruses.

Two lines of hybridomas, RT-12 and RT-14, secreting monoclonal antibodies against the reverse transcriptase from myeloblastosis-associated viruses have been prepared. The monoclonal antibodies RT-12 and RT-14 specifically react with reverse transcriptase, as has been shown by radioimmunoassay and enzyme-linked immunoassay techniques after SDS-PAGE and blotting to nitrocellulose membranes. It has been shown that antigenic determinants for RT-12 and RT-14 are stable to SDS denaturation, hence they belong to a linear type; they are located on the alpha subunit of the enzyme.

Animals↗
PubMed1987

Two exons specific for the myb proto-oncogene found upstream from the avian myeloblastosis virus-transduced myb sequences.

A partial restriction map of cloned 5.42-kb chicken DNA (clone P542, Perbal et al. 1983), covering a portion of the c-myb locus, is presented. The 5' end of the v-myb gene (approximately 0.5 kb) is located at the 3' end of P542 DNA, the remainder are the cellular sequences not transduced by avian myeloblastosis virus. Two non-contiguous DNA segments were detected within these cellular sequences which code for the 5' end of c-myb mRNA. These two exons, designated e1 and e2, are separated by a approximately equal to 1.5-kb non-coding region. Both of them are transcribed into 0.4 kb located near the 5' end of c-myb mRNA. The second exon e2 (approximately equal to 0.2 kb) is flanked at its 3' end by a short non-coding region within which virus-cell recombination took place. The possible presence of a portion of this intron in the 2.1-kb v-myb mRNA is discussed.

Animals↗
PubMed1983

Structural analysis of RNA subunits from avian myeloblastosis virus (AMV).

Subgenomic fragments were released from purified 35S AMV RNA using strongly denaturing conditions of 78% formamide and 3.9 M urea at 53 degrees C. The fragments were characterized by sedimentation and electron microscopic analysis. The presence of five distinct, reproducible size classes of RNA molecules with mean lengths 1.79 +/- 0.07 micron, 1.36 +/- 0.09 micron, 1.03 +/- 0.09 micron, 0.7 +/- 0.08 micron, and 0.42 +/- 0.09 micron were demonstrated. Analysis of poly(A)+ and poly(A)- RNA fragments revealed that splitting of 35S AMV RNA by strongly denaturing agents is not entirely random. Only one main distinct site of preferential splitting of the AMV RNA was located at 1.8 kb from the 3' end of the genome. The other three sites located at 3.2 kb, 4.5 kb, and 5.9 kb from the 3' end were much less distinct and were masked by random degradation of the molecules. The amount of poly(A)- RNA fragments of preferential lengths was increased by additional splitting of RNA molecules at preferential sites. No proteosynthetic activity was detected when subunits obtained by strong denaturation of 35S AMV RNA were analysed in a cell-free protein synthesis system.

Avian Leukosis Virus↗
PubMed1982

Studies on the structure of avian myeloblastosis virus (AMV) RNA. I. Factors affecting electron microscopic visualization of AMV RNA.

Factors affecting the visualization of single-stranded (ss) avian myeloblastosis virus (AMV) RNA by the basic protein film technique under strong denaturation conditions were studied. The basic parameters of the electron microscopic picture of ss RNA, involving the visibility, concentration and contour lengths of molecules, were found to be significantly dependent on the (1) quality of denaturing components used for full extension of AMV RNA molecules and (2) spreading and formation of the surface film. Under the conditions used, the presence of artificial secondary structures of AMV RNA molecules were dependent on batches of formamide and urea in the denaturing mixture. The spreading and formation of the surface film were affected by hydrophilicity of glass spreading ramps, speed of spreading, amount of spreading solution, amount of protein in the hyperphase, type of cytochrome c and the presence of substances influencing surface tension. The optimal conditions and spreading arrangement for visualization of ss AMV RNA are described.

Avian Leukosis Virus↗
PubMed1982

Studies on the structure of avian myeloblastosis virus (AMV) RNA. II. Integrity of the RNA in dependence on RNA isolation and virus propagation.

While sedimentation analysis revealed no basic changes in the sedimentation characteristics of 60-70 S avian myeloblastosis virus (AMV) RNA isolated under different conditions from two sources, electron microscopy exhibited differences in the length distributions of this RNA. Based on the length distribution profiles, phenol extraction showed a higher degradation effect in comparison with direct gradient centrifugation of viral lysates. Proteinase K appeared to be a more suitable nuclease inhibitor than diethylpyrocarbonate during the isolation of AMV RNA. Fundamental differences between length distributions of 60-70 S RNA from plasma AMV and that from AMV propagated in cultured leukaemic myeloblasts were found. Precipitation with ethanol had no effect on the length distribution of AMV RNA. Nor did storage of leukaemic chicken plasma at -70 degrees C and one thawing substantially influence the lengths of AMV RNA. The heterogeneous length distributions of 60-70 S AMV RNA molecules with preferential size-classes were independent of virus propagation and RNA isolation. The results support the idea of preferential splitting of AMV RNA at specific sites.

Avian Leukosis Virus↗
PubMed1982

Studies on the structure of avian myeloblastosis virus (AMV) RNA. III. Electron microscopic definition of secondary structure.

The secondary structure of avian myeloblastosis virus (AMV) RNA was characterized by electron microscopy under moderately denaturing spreading conditions. Under denaturation by aqueous 44% formamide or 77% formamide in the presence of salts, partly stretched RNA molecules with measurable double-stranded regions were observed. This approach allowed the localization from 5 to 11 regions of preserved secondary structure on AMV RNA molecules. Topographic analysis revealed a nonrandom occurrence of stable secondary structures in several prevalent regions. These regions with higher secondary structure stability revealed certain similarity to hairpin structures localized by electron microscopy on Rous sarcoma virus RNA or to highly structured regions found on this RNA by T1 ribonuclease oligonucleotide analysis.

Avian Leukosis Virus↗
PubMed1982

Electron microscopic studies on the structure of 60-70S RNA of avian myeloblastosis virus.

Structural properties of the 60-70S RNA complex of avian myeloblastosis virus (AMV) were analysed in electron microscope after treatment under a set of non-denaturing, gently and strongly denaturing conditions. By selected denaturing conditions, the significant fraction of 60-70S AMV RNA molecules revealed partially unfolded structures either in a dimer or a more complex form and in a length corresponding to mol. wt. of 5.6 X 10(6). The typical dimers contained a characteristic central structure connecting the subunits and similar to those described for Rous sarcoma virus (RSV) and mammalian retrovirus RNAs. This dimer linkage in the AMV genome occurred at 384 +/- 43 nucleotides from one end of each subunit. Besides partially unfolded complexes, collapsed structures and extended linear molecules were observed. The length of majority of the linear molecules had reached a half of that of the partially unfolded complexes corresponding to the mol. wt. of monomers estimated under conditions of strong denaturation to be 2.8 X 10(6). Based on our findings, we conclude that the genome of AMV shares the dimer structure with RSV and mammalian retroviruses. We also conclude that the secondary structure of AMV RNA molecule is more labile than that of RNA of mammalian retroviruses.

Avian Leukosis Virus↗
PubMed1982

In vitro processing of avian oncovirus precursor polypeptide Pr76gag by virion protein p15.

In vitro cleavage by p15 virion protein of the primary translation product of the avian oncovirus gag gene, the precursor polypeptide Pr76gag, was studied in kinetic experiments. Nondenatured 35S-methionine-labelled Pr76gag, which was synthetized in reticulocyte lysate programmed by genomic AMV-RNA was used as a substrate, pure native p15 (AMV) as a protease. Reaction conditions were optimal for in vitro protein synthesis. Composition of the cleavage products was estimated by immune precipitation with monospecific antisera against internal structural virion (AMV) proteins p27, p19, p15, and p12, and their size by SDS-PAGE. Monospecificity of each of the antisera was assessed by adsorption with the three respective heterologous gag proteins, followed by immune precipitation of 35S-methionine-labelled proteins of the virion (AMV) lysate. The p15-mediated in vitro cleavage proceeds rapidly and specifically. In the early stages (10 min incubation with p15) the Pr76gag was absent, and an optimum amount of six cleavage intermediates with the following size, composition (shown in parentheses) and orientation (determined form the presence or absence of antigenic determinants of p19 or p15 proteins as N- or C-terminal moieties of the precursor) was found: N-terminal fragments of 66K (p19, p27, p12) and 60K (p19, p27); internal fragments of 37K (p27, p12); C-terminal fragments of 51K (p27, p12, p15), 32K (p12, p15) and 21K (p12, p15). These intermediates were converted into the following four final cleavage products, as the only polypeptides detected after prolonged (5 h) incubations: mature p27 and mature p15 proteins and 38K and 34K polypeptides containing only p19 antigenic determinants. Mature p12 an p19 proteins were not found among cleavage products. Autocatalytic cleavage of Pr76gag was not observed. The results hav allowed us to conclude that the arrangement of the gag proteins in the Pr76gag is N-p19-(p10?)-p27-p12-p15-C and that under in vitro conditions p15 recognizes three correct cleavage sites on native Pr76gag: one located at p12-p15 junction and two on both sides of the p27 moiety, as well as aberrant cleavage sites located inside p12 and possibly also p10 moieties.

Antigens, Viral↗
PubMed1980

Translation of genomic avian myeloblastosis virus RNA in a cell-free protein synthesis system from rabbit reticulocytes.

Reaction conditions suitable for translation of genomic avian myeloblastosis virus RNA in micrococcal nuclease-pretreated reticulocyte lysates are described. The products of translation were characterized by immunoprecipitation and gel electrophoresis and compared with virus-specific products formed in host cells. Genomic viral RNA directed in a cell-free system the synthesis of precursors to viral structural proteins, namely Pr76gag and Pr180gag,pol.

Animals↗
PubMed1980

Studies in AMV DNA. I. Physical properties and sequence composition of DNA present in AMV virions (AMV DNA).

The basic physical properties of AMV DNA (labeled with 3H-thymidine) as well as sequence homology between 3H-AMV DNA and viral or host cell genomes were investigated. It was found that native AMV DNA is in part single-stranded (20% of the total DNA). Using CsCl density gradient centrifugation AMV DNA was separated into two bands with buoyant densities of 1.702 g/cm3 (L-AMV DNA) and 1.722 g/cm3 (H-AMV DNA) in the peak fractions. Corresponding melting temperatures were estimated to be 82.0 degrees C and 81.3 degrees C for L- and H-AMV DNA, respectively. Hybridization experiments revealed that AMV DNA is not complementary to the AMV genomic RNA but complementary prevalently to the sequences with low frequency of repetition and to the unique sequences of the myeloblast genome. These unique sequences, comprising about 40% of the total 3H-AMV DNA, were not detected in the nuclear DNA of leukosis-free chicken embryo fibroblasts. After infection of embryo cells with AMV the extent of hybridization in the region of unique sequences rose roughly by 20%. Renaturation of thermally denatured 3H-AMV DNA using the phenol emulsion reassociation technique was observed. The results suggest that AMV DNA is composed of a number of different special sequences of the host genome.

Animals↗
PubMed1980

Studies in AMV DNA. II. A possible origin of DNA present in AMV virions (AMV DNA).

Some properties of cytoplasmic nonmitochondrial DNA--cyt DNA--from chicken leukemic myeloblasts were determined and compared with corresponding characteristics of AMV DNA. Our results indicate that cyt DNA and AMV DNA molecules are akin with respect to their specific radioactivities, sedimentation behaviour and melting profiles. Furthermore, the partial lack of sequence reiteration of both cyt and AMV DNAs distinguished them from the nuclear myeloblast DNA. It is concluded that cyt DNA of myeloblasts represents a set of distinctive cellular sequences some of which are liberated into the extracellular environment and are found in the AMV preparations.

Animals↗
PubMed1976

Stepwise dissociation of high molecular weight avian myeloblastosis virus RNA: 30-40S RNA subunits--the best natural template-primer for viral reverse transcriptase.

Controlled disruption of 60S AMV RNA with formamide was used to prepare 50-55S and 30-40S RNAS. When the activities of these RNAs as templates for AMV reverse transcriptase were compared it was found that 50-55S RNA was 1-5 times and 30-40S RNA 2 to 3 times more active than 60S RNA. The 30-40S RNA produced by heating, instead of formamide disruption, was inactive as a template but activity was restored by addition of oligo(dT). 40% of the 4S RNA initially associated with the 60S RNA remained associated with all the RNA species obtained by formamide treatment but was lost on heating. It is concluded that this RNA acts as resident primer whereas the other 60% of the 4S RNA is less firmly bound and appears to have little or no primer activity. Removal of the less firmly bound 4S RNA increases the template activity of the viral RNA.

Avian Leukosis Virus↗
PubMed1976

The oncornavirus maturation process: quantitative correlation between morphological changes and conversion of genomic virion RNA.

Avian myeloblastosis virus (AMV) was harvested at different time intervals from chick leukemic myeloblasts, and the rate of the maturation process of AMV was estimated on the basis of morphological changes in the virions and conversion of genomic viral RNA. The change from immature virions characterized by the presence of an electronlucent center to the condensed mature form (with dense nucleoid) was accompanied by the conversion of 30-40S RNA to 60S RNA. Both processes were quantitatively defined, correlated, and found to proceed in parallel at the same speed. Early stages of maturation were characterized by a high turnover of immature to mature virions; 31-40% of the mature forms were found in 3.5-min virus harvests. The rate of this process decreased with time. The 17-hour harvests still revealed the presence of 10-15% of immature virions. The course of the maturation process is discussed.

Animals↗
PubMed1976

Stepwise transition of aggregate structure of high-molecular-weight avian myeloblastosis virus RNA. Mode of releasing of associated 4S RNA.

Mode of releasing of associated 4S RNA species was studied during a controlled transition of aggregate structure of high-molecular-weight AMV-RNA. It has been found that associated 4S RNA constitutes 2.5% of 60S AMV-RNA complex. Approximately 60% of associated 4S RNA is successively released during treatment of viral RNA with increasing formamide concentration, concomitantly with the transition of 60S RNA aggregate through 50--55S RNA intermediate into the final 30--40S RNA subunits. 40% of 4S RNA remains associated with 30--40S RNA subunits prepared by formamide treatment and can be released from them by heating. A procedure is thus provided both for the isolation of oncornaviral RNA subunit structures deprived of various partions of associated 4S RNA and for the fractionation of 4S RNA species according to their binding affinity to the genome oncornaviral RNA.

Avian Leukosis Virus↗