Micromorphology of cytoplasmic nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA.

Nucleoprotein (NP) complexes constituting the three basic components (A, B, C) of the postmicrosomal sediment (POMS) of chicken leukemic myeloblasts (CHLMs) which contain extrachromosomal DNA closely related to avian myeloblastosis virus DNA were analyzed electron microscopically. It was shown that these NP complexes resemble micromorphologically, depending on the origin of their POMS components, NP structures involved in three successive stages of early DNA synthesis. Nucleic acids harbored in these NP complexes exhibited micromorphological features typical for replicative structures. It was confirmed electron microscopically that the extrachromosomal DNA of CHLMs replicative in nature and of three length classes is organized into special NP complexes, each of which, as demonstrated, represents a unique reaction machinery of early DNA synthesis.

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.

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.

Analysis of gag-myc proteins of partially transformation-defective mutants of avian myelocytomatosis virus strain MC29 by in vitro cleavage with protease p15: indication for the presence of specific cleavage site p15 in the myc domain of fusion protein.

Gag-related proteins were immunoprecipitated from [35S]methionine-labelled cells of the PR-2 line which contained p110 protein as well as from quail cells transformed by deletion mutants 10A, 10C, and 10H of MC29 virus. Immunoprecipitates were incubated with oncoviral protease p15 and cleavage products were analyzed in SDS-PAGE. The major 56K fragment (F56) of p110 was further analyzed by tryptic peptide mapping. The results showed that except for the myc domain of p110, a portion of p27 is also present in F56. Cleavage of 100K, 95K, and 90K proteins coded by three MC29 deletion mutants resulted in major fragments 66K, 60K, and 56K, respectively. The existence of further cleavage fragments and presence of the p15 specific cleavage site in the myc domain of MC29 specific proteins is discussed.

Biological activity of proviral DNA isolated from cells infected with MAV-2(0) virus.

The ability of DNA isolated from avian myeloblastosis-associated virus MAV-2(0)-infected fibroblasts to induce the synthesis of virus upon transfection of susceptible but not resistant cells was demonstrated. The virus obtained, when inoculated into 12-day-old embryos, led to the manifestation of osteopetrosis after prolonged incubation and to the induction of nephroblastomas in some cases. Positive transfection with DNA from the non-virus producing myeloblast cell line was not detected on bone marrow cells and fibroblasts preinfected with tdB77-C virus. After transfection with DNA from virus-producing myeloblasts, the reproduction of non-transforming virus was observed. This virus did not induce myeloblastosis upon infection of chickens but induced nephroblastomas on rare occasions.

Comparison of avian MC29 and MC31 viral onc proteins.

Lysates of [35S]methionine-labelled quail cells transformed by MC31 virus were immunoprecipitated with anti-myc or anti-gag serum and analysed in SDS-PAGE. A protein with a molecular weight 110K was found in both immunoprecipitates. Thus, the product of the MC31 genome is a gag-myc fusion protein with the molecular weight as the product of the MC29 genome. Comparison by tryptic peptide mapping and p15 cleavage analysis showed no differences between both P110 proteins.

Nucleoprotein complexes harboring an extrachromosomal DNA closely related to 7 S DNA of avian myeloblastosis virus: physico-chemical properties and representation of nucleic acids.

The source of avian myeloblastosis virus (AMV) DNA, an extrachromosomal small polydisperse DNA, present in the material forming the postmicrosomal sediment (POMS) of lysed chicken leukemic myeloblasts (CHLMs) is organized into nucleoprotein (NP) complexes containing always RNA. This material, radioactively double-labelled for DNA and RNA, separated in isopycnic sucrose gradients into three POMS components (A,B,C) differing from one another in properties of labelling for DNA and RNA, sucrose densities (1.21, 1.18 and 1.08 g/cm3 for components A, B and C, respectively) and sedimentation properties of NP complexes which constituted the individual POMS components. The NP complexes present in representative fractions of POMS components A, B and C sedimented at 37.3 and 27.3, at 15.3 and 7.4, and at 11.3 and 5.5, respectively. They differed also in the length of DNAs they were harboring. Radioactively double-labelled nucleic acids (NAs) of POMS components A, B and C sedimented at 9, 7 and 3.5 S, respectively, and the sedimentation characteristics of both labels corresponded with those significant for replication intermediates. Electrophoretic characteristics of these NAs indicated that we dealt with DNA and RNA products of a lagging DNA strand synthesis (LSS) taking place, evidently, on pieces of the lagging sites of replicating DNA strands that were cut out at predicted sites by nucleases. As regards the origin of AMV DNA, we show that the major and minor portions of this DNA might be descending from NAs harbored in NP complexes of POMS components B and C, respectively, pointing out selectivity of segregation of this DNA from the cell into AMV core.

Activities of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA.

Nucleoprotein (NP) complexes constituting the material of the postmicrosomal sediment (POMS) and its three basic components (A, B, C) (Ríman and Sulová, 1997a), harboring an extrachromosomal DNA closely related to AMV DNA were found to possess DNA- and RNA-synthesizing activities (SAs) reflecting the ability of this material to be intensely labelled for DNA and RNA, respectively. The types of these NA-SAs were compatible with those significant for a lagging DNA strand synthesis (LSS). The use of selective inhibitors and of the proliferating cell nuclear antigen (PCNA) disclosed a successive involvement of alpha DNA polymerase (pol) and PCNA-insensitive delta DNA pol in LSS. In this respect, we show gradual changes in the representation of activities (As) of both mentioned DNA pols in the NP complexes of the individual POMS components. Those of POMS component C contained alpha DNA pol As only, while a distinct portion of DNA SAs of POMS component B was represented on expense of alpha DNA pol As by PCNA-insensitive delta DNA pol (epsilon DNA pol), As which represented practically all the DNA SAs of POMS component A. The type of RNA SAs of this material represented mostly by primase (Pr) As corresponded well with the nature of LSS. An exception was represented by a minor portion of RNA-SAs of POMS component A which was alpha amanitin-sensitive like RNA pol II. Moreover, analyzing this natural model replication system, we found that the carbonyldiphosphonate (COMDP), a selective inhibitor of the PCNA-insensitive delta DNA pol, was a strong activator of Pr-As and/or Pr-alpha DNA pol As of NP complexes of POMS component C.

Products of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA.

Nucleoprotein (NP) complexes present in selected fractions of the separated three basic components A, B, C of the postmicrosomal sediment (POMS) (Ríman and Sulová, 1997a) were used as a source of nucleic acid synthesizing activities (NA-SAs) expressed in reactions in vitro. These were performed in the absence and presence of N2-(p-butylphenyl) deoxyguanosine 5'-triphosphate (BuPdGTP), aphidicolin (Aph) and carbonyldiphosphonate (COMDP), inhibitors allowing differentiation between two DNA polymerases (pols) involved in a lagging DNA strand synthesis (LSS). Reaction products were isolated and analyzed by polyacrylamide gel electrophoresis (PAGE) in denaturing conditions. Products labelled with [alpha-32P]dAMP or [alpha-32P]AMP were represented by intermediates significant for LSS. Okazaki fragment precursors, whose synthesis was inhibited by BuPdGTP and resistant to Aph, and whose radioactive RNA label was DNase I-sensitive, represented products formed in vitro by NP complexes of POMS component C. Okazaki fragments 127 b in length, whose synthesis was insensitive to BuPdGTP but inhibited by Aph and COMDP, were characteristic of the reactions accomplished by NP complexes of POMS component B while products of NA-SAs of NP complexes of POMS component A were represented by Okazaki fragments up to 280 b in length, whose synthesis was most sensitive to Aph. In accord with previous data (Ríman and Sulová, 1997b), COMDP strongly stimulated production of RNAs corresponding in length with initiator RNAs (iRNAs). However, in dependence on reaction conditions also ribodeoxyribonucleotide primers can be produced by NP complexes of POMS component C, suggesting the occurrence of two primase (Pr) catalytic modes influenced by dNTP/rNTP relation and thus, by COMDP, a strong competitor for dNTPs. These results represent the first direct evidence that an extrachromosomal DNA organized into special NP complexes can be replicated extrachromosomally by a mechanism of LSS.

Okazaki fragments, a constant component of avian myeloblastosis virus core-bound 7 S DNA.

We have shown that the unusual CsCl-buoyant density and velocity sedimentation properties of the isolated host 7 S DNA species associated with the core fraction of avian myeloblastosis virus (AMV) are made mainly by tight association of RNA pieces prevalently joined to the single-stranded portion of this material. It was shown indirectly on sedimentation patterns of [methyl-3H]thymidine and [14C]uridine double-labelled and glyoxylated total AMV DNA, and directly in phosphorylation experiments with T4 polynucleotide kinase performed on the single-stranded portion of AMV DNA that the RNA-DNA link in AMV DNA is of a covalent nature and that the 5'-terminal end of DNA at the RNA-DNA junction is occupied by all four common deoxyribonucleotides. This first evidence of the presence of Okazaki fragments in 7 S AMV DNA clearly indicates that this DNA does not represent a randomly fragmented host DNA included by chance into virions but special fragments of host DNA having the properties of DNA replicative structures with possible consequences for some viral function(s) including those involved in virus-cell interactions.

Primase activities constantly present in avian myeloblastosis virus core isolates: detection and basic characteristics.

RNA-synthesizing activities (RNA-SAs) by its nature identical with primase activities (Pr-As) were found to be constantly present in avain myeloblastosis virus (AMV) core isolates. Their endogenous templates are molecules of the virus core-bound host cell DNA (AMV DNA) (Ríman and Beaudreau, 1970) that have been recently recognized as a collection of still active early replicative structures (Ríman et al., 1993b). Like the Pr-As, the RNA-SAs are not inhibited by alpha-amanitin nor by aphidicolin and they show a mutually competitive affinity for ATP and GTP. Their reaction products treated with DNase I are short RNAs similar in length to initiator RNAs (iRNAs), their precursors and degradation products. In AMV core proteins separated in isopycnic CsCl gradients, they are chiefly located in the density region of reverse transcriptase activities (RT-As) but with a distinct peak fraction. Like Pr-As, they are able to use poly(dT) as template and to form, in the presence of [alpha-32P]ATP, products that after DNase I treatment consist of poly(rA) molecules similar in length to iRNA monomers and multimers. Like the Pr-As, they are able to complement E. coli DNA polymerase (pol) I reactions. They occur in the analyzed AMV core proteins as six distinct sedimentation species (PrA-SS). This, together with other relevant properties, indicates the presence of Pr-As associated with molecules of a primase-alpha DNA polymerase enzyme complex, its degradation products and 'free' primase monomers.

Avian myeloblastosis virus core-bound 7 S DNA, a collection of minute replicative host-cell DNA structures.

The early replicative nature of avian myeloblastosis virus core-bound 7 S DNA (AMV DNA), indicated by our preceding findings (Ríman et al., 1993), has been confirmed using various experimental approaches. It has been shown by agarose and polyacrylamide gel electrophoresis that this DNA represents actually a collection of molecules the size of which is strongly reminiscent of the minute early replicative structures found in DNA of sea urchin embryos (Baldari et al., 1978). With such a characteristic correspond, the sequence properties of the individual AMV DNA clones, the majority of which were found to be AT-rich with ARS-like motifs and stretches of A-residues carrying conformational requirements for bending. In comparative hybridization experiments, AMV DNA exhibited the highest homology with chicken leukaemic myeloblast scaffold-bound DNA. Compatible with high replicative activity of AMV DNA was also found its specific [methyl-3H]thymidine radioactivity. The constancy of the virus content of this DNA and its virus age-dependent cleavage changes taking place inside the virus core structure open the question of possible significance of this special host DNA for the reaction machinery represented by the retroviral nucleoprotein core complex.

Avian myeloblastosis virus core-bound 7 S DNA, highly bent minute structures with sequence-directed curvature.

Structural properties and length distribution profile of 7 S avian myeloblastosis virus (AMV) DNA were studied by means of electron microscopy using two different techniques. This DNA represents mostly double strands, the single strands being in minority. We have shown directly that this DNA forms a bent structure typical of the majority of molecules. These bends are sensitive to the distamycin treatment which stretches most of the bent molecules. Some amount (up to 30%) of circular DNA molecules was detected also in DNA preparations, the nature and the size of which are reminiscent of electron microscopic data on microbubbles of replicating DNA. No specific AMV DNA structural features were found using osmium-tetroxide treatment. The basic size of AMV DNA was estimated to be approximately 150 bp, but its multimers were also detected. Their presence and significance is discussed.

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.

[Direct and indirect detection of Chlamydia in infertile married couples in the assisted reproduction program]. / Prímý a neprímý prukaz chlamydií u neplodných manzelských páru zarazených do programu asistované reprodukce.

The authors pay attention to the relationship of pregnancy in women enlisted in the IVF programme and the detection of Chlamydia infection. A group of 74 infertile couples were investigated. A significant relationship was found between confirmed Chlamydia infection in women and men. Moreover a significant relationship was found between Chlamydia infection confirmed by immunofluorescence in men and impregnation in women in the IVF programme. This means that in female partners of men with acute Chlamydia infection the results of IVF are less successful. The authors recommend that all couples enlisted in the IVF programme should be examined for this infection and if it is positive given adequate treatment.