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Our immune system, fine-tuned by a long evolution, has a near-infinite capacity to recognize potential pathogens and mutant self proteins. It has also got a varied arsenal of killing mechanisms to battle intruders and mutant cells. Since... more
Our immune system, fine-tuned by a long evolution, has a near-infinite capacity to recognize potential pathogens and mutant self proteins. It has also got a varied arsenal of killing mechanisms to battle intruders and mutant cells. Since malignant transformation involves 1) mutations of proteins of various classes and 2) over-expression of non-altered genes, either related or unrelated to the oncogenic process, the adaptive immune system has the potential to recognize and clear malignantly transformed cells. Immunotherapeutic interventions might 1) trigger an immune response to otherwise tolerated tumor antigens, 2) enhance the existing, but insufficient anti-tumor immune response, add new receptors, recombinant antibodies or T cell receptors, to the system, or 4) rely on the transfer of ex vivo expanded immune effector cells. Although tumor immunotherapy is decades-old, immune checkpoint inhibitor therapy, probably the most significant breakthrough, is a development of the last few...
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Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which... more
Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestio...
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Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained... more
Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%...
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We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for... more
We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two...
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ABSTRACT The gene coding for the sequence-specific modification methylase of Bacillus sphaericus R was cloned in E. coli by means of the pBR322 plasmid. Selection was based on the expression of the methylase gene which rendered the... more
ABSTRACT The gene coding for the sequence-specific modification methylase of Bacillus sphaericus R was cloned in E. coli by means of the pBR322 plasmid. Selection was based on the expression of the methylase gene which rendered the recombinant plasmid resistant against the Bsp restriction endonuclease. The functionally intact methylase gene was carried by a 9 kB BamHI, or a smaller 2, 5 kB EcoRI fragment. The strain harboring the recombinant plasmid exhibited Bsp-specific methylase activity, the enzyme appeared to be identical with that of the parental B. sphaericus strain. The recombinants did not have any Bsp restriction nuclease activity.
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The nucleotide sequence of the gene coding for the 5'-GGCC and 5'-CCGG specific DNA methyltransferase of the Bacillus subtilis phage SPR was determined by the Maxam-Gilbert procedure. Transcriptional and... more
The nucleotide sequence of the gene coding for the 5'-GGCC and 5'-CCGG specific DNA methyltransferase of the Bacillus subtilis phage SPR was determined by the Maxam-Gilbert procedure. Transcriptional and translational signals of the sequence were assigned with the help of S1 mapping and translation in E. coli minicells. The gene codes for a 49 kd polypeptide. The amino acid sequence of the SPR methylase shows regions of homology with the sequence of the 5'-GGCC-specific BspRI modification methylase.
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Research Interests: Biochemistry, Genetics, Biology, Medicine, Biological Sciences, and 15 moreDNA, Environmental Sciences, Escherichia coli, Methylation, DNA methylation, Nucleic Acids, High Pressure Liquid Chromatography, Methionine, Cysteine, Protein Conformation, Amino Acid Sequence, Methyl Group, Methyltransferase, Molecular Sequence Data, and cytosine
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Research Interests: Genetics, Molecular Biology, Biology, Medicine, Biological Sciences, and 15 moreEnvironmental Sciences, Escherichia coli, Methylation, Nucleic Acids, Gene, NAR, Enzyme, Molecular cloning, Amino Acid Sequence, Base Sequence, Methyltransferase, Molecular Sequence Data, restriction enzyme, Restriction Mapping, and Nucleotide sequence
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The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated... more
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
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Expression of the methyltransferase gene from Bacillus subtilis lysogenizing phage SP beta B was studied by analyzing the sensitivity of the hybrid plasmid DNAs to restriction by the enzymes BspRI, Hpall and Mspl. The gene produces the... more
Expression of the methyltransferase gene from Bacillus subtilis lysogenizing phage SP beta B was studied by analyzing the sensitivity of the hybrid plasmid DNAs to restriction by the enzymes BspRI, Hpall and Mspl. The gene produces the methylase M. BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B derived gene also directs the synthesis in E. coli of a second methylase activity (M. BsuP beta BII) with 5'-CCGG specificity. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene.
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1.) A SinI DNS-metiltranszferáz egyes mutánsai (N172S, V173L, N172S+V173L és L214S+Y229H) relaxált specifitást mutatnak, azaz a GGWCC szekvencia mellett a GGSCC szekvenciát is metilálják. Valamennyi eddig izolált, relaxált specifitást... more
1.) A SinI DNS-metiltranszferáz egyes mutánsai (N172S, V173L, N172S+V173L és L214S+Y229H) relaxált specifitást mutatnak, azaz a GGWCC szekvencia mellett a GGSCC szekvenciát is metilálják. Valamennyi eddig izolált, relaxált specifitást okozó mutáció az enzim feltételezett nagy doménjében található. Ez azt jelzi, hogy legalábbis a W vs. S megkülönböztetésben szerepe van a nagy domén és a kis árok közötti kölcsönhatásoknak is. Valamennyi vizsgált mutánsra jellemző a GGSCC szubsztráttal szembeni kcat érték jelentős megnövekedése. 2.) Klónoztuk két restrikciós endonukleáz (BspRI,GGCC; BepI, CGCG) génjét. A DNS szekvencia alapján a BspRI 317, a BepI 348 aminosavból áll. A gének átalakításával és expressziós vektorba ültetésével túltermelő klónokat hoztunk létre. 3.) Az SssI metiltranszferáz különböző variánsait állítottuk elő abból a célból, hogy terminálisan kovalens kötéssel oligonukleotidhoz kapcsolható legyen. A nemzetközi együttműködésben végzett munka célja egy ?programozható? speci...
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Methylation of specific cytosines in the DNA is generally believed to play some role in the regulation of gene expression in eukaryotes. However, some eukaryotes, such as Drosophila and yeast (S. Hattman, personal communication) seem not... more
Methylation of specific cytosines in the DNA is generally believed to play some role in the regulation of gene expression in eukaryotes. However, some eukaryotes, such as Drosophila and yeast (S. Hattman, personal communication) seem not to contain 5-methylcytosine in their DNA. It would be interesting to test, how gene expression in such organisms would respond to the methylation of specific cytosines in the genome. As a first step towards this goal, we have introduced the gene encoding the Bacillus sphaericus R modification methylase, which methylates the internal cytosine within the recognition sequence 5'-GGCC, into yeast cells. Southern-type hybridization to DNAs isolated from the transformed yeast clones revealed that the yeast plasmid carrying the prokaryotic methylase gene, as well as the two chromosomal genes tested (his3 and leu2) were methylated, whereas the bulk of the yeast DNA remained largely unmethylated. This indicates that the Bacillus sphaericus modification methylase was expressed in yeast but it modified only certain parts of the yeast DNA.
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Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in... more
Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non-cognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.
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A comparative thermogravimetric study of the deammoniation of NH4-mordenite, NH4-chabazite, and NH4-stilbite in oxidizing and inert atmosphere has been carried out. The results suggest that in oxygen atmosphere an intracrystalline... more
A comparative thermogravimetric study of the deammoniation of NH4-mordenite, NH4-chabazite, and NH4-stilbite in oxidizing and inert atmosphere has been carried out. The results suggest that in oxygen atmosphere an intracrystalline oxidation of thermally-excited ammonium lattice cations occurs. This reaction proceeds at much lower temperatures than the thermal deammoniation in an inert atmosphere and consequently results in a much better separation