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PubMedDate not supplied

Silencing of gene expression: implications for design of retrovirus vectors.

Transcriptional silencing of retroviruses poses a major obstacle to their use as gene therapy vectors. Silencing is most pronounced in stem cells which are desirable targets for therapeutic gene delivery. Many vector designs combat silencing through cis-modifications of retroviral vector sequences. These designs include mutations of known retroviral silencer elements, addition of positive regulatory elements and insulator elements to protect the transgene from negative position effects. Similar strategies are being applied to lentiviral vectors that readily infect non-dividing quiescent stem cells. Collectively these cis-modifications have significantly improved vector design but optimal expression may require additional intervention to escape completely the trans-factors that scan for foreign DNA, establish silencing in stem cells and maintain silencing in their progeny. Cytosine methylation of CpG sites was proposed to cause retroviral silencing over 20 years ago. However, several studies provide evidence that retrovirus silencing acts through methylase-independent mechanisms. We propose an alternative silencing mechanism initiated by a speculative stem cell-specific "somno-complex". Further understanding of retroviral silencing mechanisms will facilitate better gene therapy vector design and raise new strategies to block transcriptional silencing in transduced stem cells.

Chromatin↗
PubMed2005

Methylation of MRE11 regulates its nuclear compartmentalization.

The cellular response to DNA damage includes the orderly recruitment of many protein complexes to DNA lesions. The MRE11-RAD50-NBS1 (MRN) complex is well known to localize early to sites of DNA damage, but the post-translational modifications required to mobilize it to DNA damage sites are poorly understood. Recently, we have shown that MRE11 is arginine methylated in a C-terminal glycine-arginine rich (GAR) domain by protein arginine methyltransferase 1 (PRMT1). Arginine methylation is required for the exonuclease activity of MRE11 and the intra-S phase DNA damage response. Herein, we report that cells treated with methylase inhibitors failed to relocalize MRE11 from PML nuclear bodies to sites of DNA damage and formed few gamma-H2AX foci. We also demonstrate that PRMT1 is a component of PML nuclear bodies where it colocalizes with MRE11.Using cellular fractionation, we demonstrate that methylated MRE11 is predominantly associated with nuclear structures and that MRE11 methylated arginines were required for this association. These results suggest that MRE11 methylation regulates its association with nuclear structures such as PML nuclear bodies and sites of DNA damage.

Arginine↗
PubMed1997

Peculiarities of gene expression of the EcoRII modification-restriction system.

The restriction-modification genes of the EcoRII system have been cloned into plasmids under control of phage-specific promoters T7 and SP6. The transcription was induced by cell infection with the recombinant M13 phages with the corresponding genes of phage RNA-polymerases under control of the Plac-promoter in the presence of IPTG. The induction yields significant amounts of EcoRII DNA-methylase for both phage-specific promoters. In both cases no increase in EcoRII endonuclease expression could be achieved. We hypothesize that the expression of the endonuclease gene is regulated on the translational level.

Bacteriophage M13↗
PubMed1988

Cloning and complete nucleotide sequences of the type II restriction-modification genes of Salmonella infantis.

The complete type II restriction-modification system of Salmonella infantis was cloned in Escherichia coli as an R . Sau3AI fragment of 3,430 base pairs. The clone was shown to express the restriction endonuclease as well as the modification methylase. The nucleotide sequence of the above fragment showed two open reading frames of 461 and 230 codons in tail-to-tail orientation. These were shown to represent the modification methylase M . SinI and the restriction endonuclease R . SinI, respectively. The methylase M . SinI amino acid sequence revealed a considerable similarity to those of other deoxycytidylate methylases. In contrast, endonuclease R . SinI did not exhibit such a similarity to other restriction enzymes.

Amino Acids↗
PubMed1980

Restriction and modification in Bacillus subtilis: identification of a gene in the temperate phage SP beta coding for a BsuR specific modification methyltransferase.

A gene coding for a modifying DNA-methyltransferase which methylates the central C in the BsuR recognition sequence 5'GGCC was identified in the genome of the temperature Bacillus subtilis phage SP beta. This gene is expressed only after induction of the prophage by either mitomycin C or UV. The presence of active methyltransferase in induced cells leads to modification of BsuR recognition sites in SP beta DNA as well as in heterologous DNA.

Bacillus subtilis↗
PubMed2000

Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes.

Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.

Bacteriophages↗
PubMed1988

Cloning the FnuDI, NaeI, NcoI and XbaI restriction-modification systems.

Methyltransferase genes from the FnuDI, NaeI, NcoI, and XbaI restriction-modification systems have been isolated in Escherichia coli by 'shot-gun' cloning bacterial DNA fragments into plasmid vectors and selecting for protectively modified molecules that resist digestion by the corresponding restriction endonuclease.

Cloning, Molecular↗
PubMed1978

Purification and properties of a new restriction endonuclease from Haemophilus influenzae Rf.

Haemophilus influenzae Rf 232, showing the phenomena of restriction and modification, contains an endonuclease that inactivates in vitro the biological activity of DNAs lacking the strain-specific modification. This specific restriction endonuclease has been purified to near homogeneity by a procedure that includes DNA-agarose chromatography. This highly purified enzyme requires ATP and Mg2+ for activity and is stimulated by S-adenosylmethionine. The enzyme seems to cleave DNA at well-defined sites, since it produces a specific pattern of bands upon agarose gel electrophoresis. The enzyme has no ATPase activity. A methylase activity is observed in the course of the endonucleolytic reaction, which probably protects some of the DNA sites from cleavage.

Adenosine Triphosphatases↗
PubMed2005

DNA methylating and demethylating treatments modify phenotype and cell wall differentiation state in sugarbeet cell lines.

In plants organogenesis, cell differentiation and dedifferentiation are fundamental processes allowing high developmental plasticity. Such plasticity involved epigenetic mechanisms but limited knowledge is available concerning quantitative aspects. Three sugarbeet (Beta vulgaris L. altissima) cell lines originating from the same mother plant and exhibiting graduate states of morphogenesis were used to assess whether these differences could be related or not to changes in DNA methylation levels. Methylcytosine percentages from 18.3 to 28.8% and distinct levels of DNA methyltransferase (EC 2.1.1.37) activities were shown in the three cell lines. The lowest methylcytosine percentage was associated to organogenesis. In order to test the plasticity of these cell lines, various treatments causing DNA hypo or hypermethylation were performed at different times and concentrations. In this collection of treated lines with+/-10% of methylcytosine percentages, loss of organogenic properties and cell dedifferentiation were observed. As cell wall formation fits well with cell differentiation state, the lignification process was further investigated in treated and untreated lines as a biochemical marker of the phenotypic changes. For example, peroxidase specific activities (EC 1.11.1.7) varied from 0.7 to 0.02 pkat mg(-1) of protein in organogenic and dedifferentiated lines, respectively. A negative relationship between peroxidase activities, incorporation of cell wall-bound phenolic compounds as ferulate and sinapate derivatives and methylcytosine percentages was obtained. This is the first biochemical evidence that phenotypic changes in plant cells induced by DNA hypo- or hypermethylating treatments are correlated in a linear relationship to modifications of the cell wall differentiation state.

5-Methylcytosine↗
PubMed1992

Type II DNA restriction-modification system and an endonuclease from the ruminal bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II restriction endonuclease, FsuI, which recognizes the sequence 5'-GG(A/T)CC-3'. Analysis of the restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 5' protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II restriction-modification system of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium.

Base Sequence↗
PubMed1993

[Hydrolysis of DNA-duplexes, containing 5-fluorodeoxycytidine by restriction endonucleases].

Cleavage by restriction endonucleases MvaI kappa EcoRII of DNA duplexes, in which the internal deoxycytidine in one of the strands of the recognition site is substituted for 5-fluorodeoxycytidine, has been studied. It has been found that the modified strands of these duplexes are scissed by endonuclease MvaI with a greater efficiency than the intact ones. Endonuclease EcoRII does not practically cleave such substrate analogs. The UV absorbance spectra of 5-fluorodeoxycytidine and the 14-member oligodeoxyribonucleotide with modification in the middle of the strand have been measured. A hypothesis has been put forward about the structural features of 5-fluorodeoxycytidine-containing DNA duplexes.

Base Sequence↗
PubMed1975

X inactivation, differentiation, and DNA methylation.

A model based on DNA methylation is proposed to explain the initiation and maintenance of mammalian X inactivation and certain aspects of other permanent events in eukaryotic cell differentiation. A key feature of the model is the proposal of sequence-specific DNA methylases that methylate unmethylated sites with great difficulty but easily methylate half-methylated sites. Although such enzymes have not yet been detected in eukaryotes, they are known in bacteria. An argument is presented, based on recent data on DNA-binding proteins, that DNA methylation should affect the binding of regulatory proteins. In support of the model, short reviews are included covering both mammalian X inactivation and bacterial restriction and modification enzymes.

Animals↗
PubMed1981

Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K.

The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+. The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E. coli K with equal efficiency. In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor. Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation. The rate of enzyme activation was determined using the AdoMet analog Sinefungin. The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions.

Bacteriophage lambda↗
PubMed2002

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system.

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.

DNA Restriction-Modification Enzymes↗
PubMed1985

Investigation of restriction-modification enzymes from M. varians RFL19 with a new type of specificity toward modification of substrate.

The characterization of MvaI restriction-modification enzymes, isolated from Micrococcus varians RFL19, is reported. Both enzymes recognize the 5'CC decreases (A/T)GG nucleotide sequence. The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N4-methylcytosine. This type of modification protects the substrate from R.MvaI cleavage. 5-Methylcytosine in the same position of the recognition sequence does not protect the substrate from R.MvaI cleavage. R.MvaI proved to be the first example of a restriction endonuclease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence. M.MvaI modifies DNA dcm+ in vitro yielding N4,5-dimethylcytosine. N4-methylcytosine cannot be differentiated from cytosine using the Maxam-Gilbert DNA sequencing procedure.

5-Methylcytosine↗
PubMed1985

Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli.

A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.

Bacteriophage lambda↗
PubMed1997

Nucleotide sequences and gene organization of TaqI endonuclease isoschizomers from Thermus sp. SM32 and Thermus filiformis Tok6A1.

Eight TaqI isoschizomer genes, two from Yellowstone National Park, one from Japan, two from New Zealand, two from Portugal, and one from the Azores (1000 miles west of Portugal), were PCR-amplified and sequenced. Sequence alignment of isoschizomers isolated from close geographical locations shows identical or almost identical protein sequences, while isoschizomers from distant sites demonstrate considerable diversity, ranging from 54 to 75% in amino acid identity. Accordingly, these isoschizomers were arranged into four geographical groups, i.e., USA as represented by Thermus aquaticus YT1, Japan by Thermus thermophilus HB8, New Zealand by Thermus filiformis Tok6A1, Portugal by Thermus sp. SM32. The complete ORFs of two new representative genes, tfiTok6A1I and tsp32IR, were obtained by bubble PCR. Unlike M . TaqI-R.TaqI and M . TthHB8I-R . TthHB8I which exhibit an unusual 13-codon overlap, the methylase and endonuclease genes are each separated by 15 nucleotides in the TfiTok6A1I and Tsp32IR restriction-modification systems. Phylogenetic analysis suggests that initially TfiTok6A1I diverged from a common ancestor, then Tsp32IR branched out, and finally TaqI and TthHB8I diverged from each other during evolution.

Amino Acid Sequence↗