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M Trávnícek

Publications and source records attributed to M Trávnícek.

At least 19 recordsLinked to original sources
PubMed1992

Isolation, biochemical characterization and crystallization of the p15gag proteinase of myeloblastosis associated virus expressed in E. coli.

1. The p15gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (Pr25lac-delta gag) was accompanied by its structurally correct processing. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N-terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 3. Contrary to reported data, the amino acid sequence of the p15gag proteinase differs from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well as 2.3 A without deterioration.

Amino Acid Sequence↗
PubMed1990

Inhibition of avian myeloblastosis virus reverse transcriptase by diphosphates of acyclic phosphonylmethyl nucleotide analogues.

Diphosphates of N-(2-phosphonylmethoxyethyl) derivatives of heterocyclic bases were studied in the endogenous oligo(dT)12-18 primed reaction of reverse transcriptase from detergent-disrupted AMV(MAV) retrovirions. These diphosphates (analogues of nucleotide 5'-triphosphates) exhibited an inhibitory activity towards reverse transcriptase. This inhibitory activity was dependent on the character of the heterocyclic base and decreased in the order: 2-aminoadenine greater than adenine greater than guanine much greater than cytosine much greater than thymine greater than uracil. The 2-aminoadenine derivative was more potent than either AZT-TP or ddTTP, while PMEApp had approximately the same potency as the two reference compounds (IC50 approximately 1 microM at 20 microM competing substrate). This finding is consistent with the antiviral activity of the parent nucleotide analogues against retroviruses (including HIV).

Adenine↗
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↗
PubMed1990

[Detection of Chlamydia trachomatis in clinical material 1985-1988].

Investigation of 529 smears from the uterine cervix of women before delivery, during the puerperium and before induced abortion revealed positive findings in 25.2% of women before delivery and in 19.3% of women before induced abortion. Perinatal infection was confirmed by a positive finding of Ch. t. in 12.8% neonates with conjunctivitis and 15.4% from the nasopharynx. The direct immunofluorescent method was used. In another group of women examined by the serological method ELISA, from a total of 182 sera in 41.7% the presence of IgG antibodies was detected. In cervical smears from 10 seropositive women in seven Ch. t. was positive.

Abortion, Induced↗
PubMed1989

A search for c-myb gene regulatory sequences: cloning and restriction analysis of the 18-kb BamHI fragment of chicken chromosomal DNA, containing the 5' part of the c-myb gene.

The 18-kb BamHI fragment of the chicken chromosomal DNA derived from the 5' end of the myb proto-oncogene has been cloned. The 5' part of this clone represented by a 7.2-kb BamHI-EcoRI fragment has been analysed by means of restriction mapping, which revealed the existence of about 2.5 kb of the CpG dinucleotide-rich sequence within this fragment. A short probe prepared from the CpG-rich sequence hybridizes with the 3.6-kb c-myb mRNA. Based on our results and published c-myb cDNA sequence, we conclude that the cloned fragment contains c-myb promoter.

Animals↗
PubMed1988

Nucleotide sequence of chicken myb proto-oncogene promoter region: detection of an evolutionarily conserved element.

The nucleotide sequence of the chicken myb proto-oncogene putative promoter region was determined and compared with the corresponding sequence of the mouse c-myb gene (1). 118 bp upstream from the initiation codon suggested by Gerondakis and Bishop (2) for the chicken c-myb protein, a 124-bp-long conserved element was found (92% identity in chicken and mouse sequences). Sequences homologous to this element were detected on Southern blots of restricted genomic DNAs from mouse, man, lizard, frog, and carp. No hybridization was observed with Drosophila, yeast, or Escherichia coli DNA. In human DNA, sequences homologous to this element were located at the 5' end of the c-myb gene, i.e. in the same position as in the chicken and mouse genes. Several lines of evidence suggest that the element is not a coding exon of a gene overlapping the c-myb gene. It may be of importance that one of the DNase I-sensitive sites and several c-myb mRNA cap sites localized recently in the mouse c-myb gene (3,4) lie within this region. It is suggested that this evolutionarily conserved element is involved in the regulation of myb proto-oncogene expression in vertebrates.

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↗
PubMed1988

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. I. Construction of MAV-1 and MAV-2 proviral restriction maps and preparation of specific proviral molecular subclones.

A 9.8 kb DNA fragment containing the complete MAV-1 provirus was recloned from the recombinant bacteriophage lambda 311411 (Perbal et al., 1985) into the plasmid pAT153. A detailed and precise restriction map of the obtained clone (pAT-MAV-1) was constructed. From compilation of this map and the known sequence of a variable portion of the MAV-2 env gene was a restriction map of MAV-2 deduced. Knowledge of the detailed pAT-MAV-1 map facilitated the preparation of five specific proviral subclones: pAT-U3 and pUC-U3 (both contain the U3 domain of the proviral LTR, which is MAV-specific and displays no homology with other hitherto known retroviruses including avian endogenous proviruses), pUC-RU5 (containing the R and U5 domains of the proviral LTR), pUC-UT5 (containing untranslated sequences flanking the 5' LTR), and pUC-UT3 (containing untranslated sequences flanking the 3' LTR). Thus tools for analysis of integrated MAV-2 proviruses in nephroblastomas induced by this virus were formed.

Animals↗
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↗
PubMed1988

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. III. Presence of defective MAV-2 proviruses in tumour DNA.

Using Southern hybridization with a series of probes derived from the MAV provirus we searched for structurally changed MAV-2 proviruses in the DNA from MAV-2-induced nephroblastomas. Anomalous fragments of MAV-2 provirus were found in all samples analysed in more detail. Comparison of the sizes of anomalous fragments detected by different probes in the same sample indicates that the fragments, at least in a majority of cases, do not represent recombinant proviruses (which might contain transduced oncogene) but deleted proviruses only. Various types of proviral deletions occur in different nephroblastoma clones, and no preferred type of deletion was found. The defects in MAV-2 sequences appear in infected chickens de novo, because defective proviruses also occur in tumours of chickens that have been infected with the plaque-purified MAV-2 preparations and, in addition, most of the proviral defects found prevent the virus from replicating and from being transmitted by infection. The regular occurrence of defective proviruses in tumour DNA supports the concept that the proviral defects are involved in oncogenesis. Hypotheses on the substance of this involvement are discussed.

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↗
PubMed1984

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.

Animals↗
PubMed1984

Effect of hemin on poly (A)-containing RNA synthesis and transport from nucleus into cytoplasm in Friend cells of the Fw line.

The effect of hemin on the transcription and post-transcriptional events (processing and transport of globin mRNA and other poly(A)-containing RNAs to the cytoplasm) was investigated in murine erythroleukemia Friend cells (MELC) of the Fw line. Uninduced Fw cells or Fw cells induced to differentiation by butyric acid were incubated in vitro without or with exogenous hemin. Short labelling periods (5-20 min) with (3H) uridine were used. The synthesis of globin mRNA and other poly(A)-containing RNAs at the level of their precursors in the nucleus and at the level of mature forms in the cytoplasm was measured. Our results indicate that hemin mainly affects the processing or transport of globin mRNA and other poly(A)-containing RNAs from the nucleus to the cytoplasm. The effect of exogenous hemin is quite different in uninduced and induced Fw cells. Exogenous hemin stimulates accumulation of poly(A)-containing RNA in nuclei of uninduced Fw cells. On the other hand exogenous hemin causes an increase in cytoplasmic globin mRNA and other cytoplasmic poly(A)-containing RNA in Fw cells induced by sodium butyrate.

Animals↗