Biomedical subjects

J Ríman

Publications and source records attributed to J Ríman.

At least 19 recordsLinked to original sources

PubMed2002

The deoxyribo-mode expression of primase activities of the primase-alpha DNA polymerase enzyme complex associated with nucleoprotein complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus core-bound DNA: influencing by carbonyldiphosphonate, mimosine and butylphenyl deoxyguanosine-5'-triphosphate.

The deoxyribo-mode expression of primase (Pr) activities of the Pr-alpha DNA polymerase (pol) enzyme complex belonging to the naturally occurring nucleoprotein (NP) complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus (AMV) core-bound DNA (Ríman and Sulová, Acta Virol. 41, 181-192 (1997)) is similarly influenced by carbonyldiphosphonate (COMDP) as the ribo-mode expression of Pr activities (Ríman, Acta Virol. 45, 109-124 (2001)). In the presence of all four common dNTPs only and dNTPs and rNTPs in the reaction medium, COMDP strongly activates the deoxyribo-mode of Pr activities and again induces a unique phenomenon of primer accumulation. These primers labeled for DNA are up to 90% alkali-resistant and sensitive to DNase I treatment. This suggests that they are constituted mostly of deoxynucleotides (dnts). In contrast to the stimulation of the ribo-mode expression of Pr activities by COMDP, the incorporated radioactivity is in this case more than one order lesser. 1-Mimosine-(alpha-amino-beta-[N-(3-hydroxy-4-pyridone)]-propionic acid (MIMO) is again able to substantially eliminate the phenomenon of primer accumulation, suggesting that also in this case the effects of COMDP and MIMO are, at certain reaction conditions, mutually exclusive and that both agents compete for the same active site responsible for mutual coupling of Pr and alpha DNA pol activities.

Avian Myeloblastosis Virus↗

PubMed2001

A DNA polymerase epsilon inhibitor activates the ribo and deoxyribo modes of primase expression and induces a unique phenomenon of primer accumulation.

Carbonyldiphosphonate (COMDP), a selective inhibitor of DNA polymerase (pol) epsilon, strongly stimulates expression of the ribo and deoxyribo modes of primase (Pr) activities of the Pr-DNA pol alpha enzyme complex associated with special cytoplasmic nucleoprotein complexes of chicken leukemic myeloblasts [J. Ríman and A. Sulová, Acta Virol. 41 (1997) 181-214]. Besides stimulation, COMDP uncouples the Pr activities from those of DNA pol alpha, inducing in this way a unique phenomenon of accumulation of primers of basic length. In the presence of dNTPs, the COMDP effect is counteracted by excess of mimosine. The mutually exclusive effects of these agents are discussed.

DNA Polymerase I↗

PubMed2001

Ori-somes, nucleoprotein complexes descending from origin regions of animal chromosomal DNA replication. A micromorphological study.

Micromorphology of nucleoprotein (NP) complexes designated according to their descent and shape as Ori-somes is presented. These NP complexes of three different types harbor molecules of cytoplasmic "small" polydisperse DNA, which descend from origin regions of chromosomal DNA replication and are equipped, as shown previously, with early DNA-synthesizing activities. By negative staining the Ori-somes are visualized as particles of irregular shape, sometimes of a subunit-like structure. Micromorphological differences in size and structural compactness noted among individual Ori-somes are dependent on their type similarly as earlier shown physico-chemically and biochemically. Such differences were also confirmed by two different spreading techniques. The most unravelled structures with electron diffuse centers belong to Ori-somes of component B associated with most active DNA synthesis. In contrast, the Ori-somes of components A and C, associated with pronounced RNA synthesis, revealed large electron-dense centers. The incidence of replicative structures present in Ori-somes corresponds with the level of their DNA-synthesizing activities.

Animals↗

PubMed2001

Anatomy of the primase-alpha DNA polymerase reaction accomplished by nucleoprotein complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus core-bound DNA: influencing by carbonyldiphosphonate, mimosine and butylphenyl deoxyguanosine-5'-triphosphate.

Activities of the primase (Pr)-alpha DNA polymerase (pol) enzyme complex belonging to the naturally occurring reaction systems represented by special NP complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus (AMV) core-bound DNA (J. Riman, A. Sulová and K. Horská, Acta virol. 39, 149-159 (1995); J. Ríman and A. Sulová, Acta virol. 41, 181-192 (1997)) were studied in the absence and presence of carbonyldiphosphonate (COMDP), mimosine (MIMO), to it related ciclopirox olamine (CPX) and butylphenyl deoxyguanosine-5'-triphosphate (BuPdGTP). Reaction products radioactively labeled for RNA and DNA and synthesized with the common four ribonucleoside triphosphates (rNTPs) or rNTPs and deoxyribonucleoside triphosphates (dNTPs) in the reaction medium, were analyzed by polyacrylamide gel electrophoresis (PAGE) at denaturing conditions. It was shown that COMDP strongly activates the Pr and uncouples its activity from that of alpha DNA pol with accumulation of initiator (i) RNAs of the basic length. This phenomenon is not affected by BuPdGTP. MIMO, in contrast, stimulates both pol activities of this enzyme complex and preserves their mutual coupling. The effects of COMDP, MIMO and CPX seem to be modulated by concentration of the ambient dNTPs. Addition of dNTPs to rNTPs makes the effects of COMDP and MIMO mutually exclusive, suggesting that both these agents, though chemically quite different, are competing for one active site responsible for coupling these both pol activities into the one Pr-alpha DNA pol reaction.

Avian Myeloblastosis Virus↗

PubMed1999

The "small" polydisperse cytoplasmic extrachromosomal DNA of chicken leukaemic myeloblasts and the avian myeloblastosis virus core-bound DNA seem to descend from origin regions of chromosomal DNA replication.

Nucleotide sequences are presented for 12, 7 and 12 cloned extrachromosomal DNAs by nature harbored in nucleoprotein (NP) complexes forming chicken leukaemic myeloblast (CHLM) post microsomal sediment (POMS) components A, B and C, respectively, and for 11 cloned avian myeloblastosis virus (AMV) DNAs. Analysis of the abundance of sequence motifs significant for eukaryotic chromosomal DNA replication origin (ori) regions (and their initiation zones) has shown that these DNAs are reminiscent of cell DNA fragments enriched in ori sequences (Rao et al., 1990) and/or sequence features of several eukaryotic chromosomal oris containing clusters of modular sequence elements (Dobbs et al., 1994). Accordingly, these DNAs, with an (A + T) content prevalently higher than that of the total cell DNA, revealed the presence of asymmetrically distributed (A + T)-rich stretches, scaffold attachment region (SAR) T consensuses, polypyrimidine nucleotide (poly(Py)) tracts and minimal Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, in abundance comparable with that of these sequences of DNA fragments enriched in oris. All these DNAs were found to be enriched also in sequence elements held as primase (Pr) attachment sites. Moreover, DNAs of POMS component B and those of AMV DNA were found to be enriched in the asymmetric pyrimidine (Py) heptanucleotide motif of Waltz et al. (1996) occurring in the initiation zones of ori region. Consequently, these extrachromosomal DNAs, portion of which represents a precursor of AMV DNA, seem to descent from initiation zones of various ori regions of an early replicating chromosomal myeloblast DNA. In addition, a possible explanation of the inclination of these DNAs to form multimers is presented.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Aphidicolin↗

PubMed1997

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.

Aphidicolin↗

PubMed1995

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1990

Binding properties of avian retroviral proteins. I. Preparation and basic characterization of ASLV NC(p12) and MA(p19).

Using SP-Sephadex column chromatography we isolated from an avian retrovirus, AMV(MAV), nucleic acid-binding proteins ASLV NC(p12) and MA(p19). As shown by several criteria, namely SDS-PAGE, PR(p15) protease activity, and nucleic acid binding assay with the use of both ss and ds DNAs, our NC(p12) and MA(p19) isolates are virtually pure proteins mutually not cross-contaminated. Rabbit anti-NC(p12) and anti-MA(p19) sera which we prepared did not cross-react mutually. We conclude that both NC(p12) and MA(p19) and antibodies against them are adequately pure preparations for investigating their nucleic acid binding specificities towards AMV(MAV) genomic RNA and MAV-1 proviral DNA using electron microscopy supported by computer analysis of electron micrographs.

Animals↗

PubMed1990

Binding properties of avian retroviral proteins. II. Binding of protein ASLV NC(p12) to viral RNA and proviral DNA.

Binding of the major avian retroviral nucleocapsid protein ASLV NC(p12) to the MAV-1 (myeloblastosis-associated virus) proviral dsDNA and viral ssRNA was analysed using electron microscopy. Specificity of the binding was estimated by special computer programs. The NC(p12) protein bound to MAV-1 proviral dsDNA (clone pAT153--MAV-1), but specificity of this binding was not found by computer evaluation. NC(p12) also bound to nondenatured 70S viral RNA at a rate of 25 +/- 3 molecules per RNA molecule. When this RNA was denatured either before or after the complexing, it showed no binding affinity for the protein. This result implies that preserved secondary structure of the viral RNA was required for the binding.

Animals↗

PubMed1990

Binding properties of avian retroviral proteins. III. Binding of protein ASLV MA(p19) to viral RNA and proviral DNA.

The binding of the avian retroviral matrix protein ASLV MA(p19) to homologous viral ssRNA and proviral dsDNA was analysed using electron microscopic methods combined with a special computer evaluation. No binding affinity of MA(p19) to homologous nondenatured or denatured viral RNA was found. In contrast, ASLV MA(p19) was shown to have one specific binding site on MAV-1 proviral dsDNA at position 6795 +/- 345 bp from the 5' end of the molecule. A second specific binding site was found in a cellular sequence.

Animals↗

PubMed1989

Transcription of the chicken myb proto-oncogene starts within a CpG island.

The nucleotide sequence of an 8.2-kb DNA fragment from the 5' proximal part of the chicken myb proto-oncogene spanning 1761 nucleotides upstream and 6436 nucleotides downstream from a presumed c-myb initiation codon was determined. A 3.3-kb G + C-rich region found in this sequence had also other features characterizing CpG islands, i.e. no CpG underrepresentation and lack of CpG methylation. In haematopoietic tissues c-myb mRNA synthesis starts in two major regions of the CpG island, namely 98 to 108 and 143 to 145 nucleotides upstream from the c-myb initiation codon. These two regions are in or close to the 124-bp evolutionarily conserved element located in the middle part of the CpG island. No alternative splicing of the 5' end of c-myb mRNA suggested earlier (1,2) was observed. The c-myb promoter contains neither TATA nor CAAT box-like structures at the usual positions. Instead, numerous potential Sp1 factor binding sites were found both upstream and downstream from the transcription initiation sites. Moreover, consensus v-myb protein DNA-binding sites were revealed in the promoter region and in sequences downstream from it.

Animals↗

PubMed1988

Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E. coli system expressing a recombinant precursor (Pr25lac-delta gag).

Processing proteases of avian and mammalian retroviruses cut the polyprotein precursors encoded by the retroviral genes into mature functional proteins. Retroviral processing proteases are still a rather poorly characterized group as to their relation to other proteases, specificity, and mechanism of enzymatic action. In avian retroviruses the generation of the processing protease itself comprises a processing cleavage event - the protease p15gag is cut off the carboxy-terminus of a gag polyprotein precursor, Pr76gag. We report here that direct and efficient production of the avian retrovirus processing protease p15gag (required for structure-function studies and rational design of inhibitors) was obtained in an E. coli system, where massive expression of a size-reduced, recombinant precursor (Pr25lac-delta gag) was accompanied by its structurally accurate processing.

Amino Acid Sequence↗