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PubMed1990

Cloning and characterization of two tandemly arranged DNA methyltransferase genes of Neisseria lactamica: an adenine-specific M.NlaIII and a cytosine-type methylase.

The gene encoding the Neisseria lactamica III DNA methyltransferase (M.NlaIII) which recognizes the sequence CATG has been cloned and expressed in Escherichia coli. DNA sequencing of a 3.125 kb EcoRI-PstI fragment localizes the M. NlaIII gene to a 334 codon open reading frame (ORF) and identifies, 468 bp downstream, a second ORF of 313 amino acids, which is referred to as M.NlaX. Both proteins are detectable in the E. coli coupled in vitro transcription-translation system; they are apparently expressed from separate N. lactamica promoters. The N-terminal half of the previously characterized M.FokI, which methylates adenine in one of the DNA strands with its asymmetric recognition sequence (GGATG), is found to have 41% sequence identity and a further 11.7% sequence similarity with M.NlaIII. Among the conserved amino acids is the wellknown DPPY sequence motif. With one exception, analysis of the nucleotides coding for the DP dipeptide in all known DPPY sequences shows the presence of an inherent DNA adenine methylation (dam) recognition site of GATC. A low level of expression of M.NlaX in E. coli prevents the elucidation of its sequence recognition specificity. Sequence analysis of M.NlaX shows that it is closely related to the group of monospecific 5-methylcytosine DNA methyltransferases (M.EcoRII, Dcm, M.HpaII and M.HhaI) which all have a modified cytosine at the second position of the recognition sequences. Both M.EcoRII and Dcm amino acid sequences are about 50% identical with M.NlaX; a considerable degree of sequence identity is found in the so-called variable region which is believed to be responsible for sequence recognition specificity. M.NlaX is probably the counterpart to the E. coli Dcm in N. lactamica.

Amino Acid Sequence↗
PubMed1979

Studies of viable T4 bacteriophage containing cytosine-substituted DNA (T4dC phage). II. Cleavage of T4dC DNA by endonuclease SalI and bam HI.

Digestion of non-glucosylated and cytosine-substituted T4 phage (T4dC) DNA with SalI restriction endonuclease showed that the DNA had nine SalI-sensitive sites. There were eight SalI sites in DNA from a strain which had a deletion in the rII-denB-ndd region. The comparison of two digestion patterns indicated that one of the SalI-sensitive sites was present in the deleted region and that the SalI-F fragments (8.4 x 10(6) daltons) was located adjacent to the SalI-C or SalI-D fragments (15.5 x 10(6) daltons) on the T4 chromosome. The DNA gave no detectable cleavage product when digested with BamHI endonuclease alone, while, when digested successively with BamHI and SalI, the DNA yielded two new digestion products in place of one fragment formed by SalI alone. The BamHI-sensitive site was in the SalI-A fragment (25.2 x 10(6) daltons). The usefulness of this information for making cleavage maps of T4 phage chromosome is discussed.

Coliphages↗
PubMed1996

Identification of the gene encoding the DNA (cytosine-5) methyltransferase of lymphocystis disease virus.

The gene encoding the DNA (cytosine-5) methyltransferase (m5C-MTase) of lymphocystis disease virus (flounder isolate, LCDV-1) has been identified by polymerase chain reaction (PCR) using oligonucleotide primers synthesized corresponding to different regions of the m5C-MTase gene of frog virus 3 (FV3). A DNA fragment of 487 bp was amplified using oligonucleotide primers L3 and R4 which correspond to the nucleotide positions 87 to 109 and 530 to 550 of the m5C-MTase gene of FV3, respectively. The DNA nucleotide sequence of the PCR product was determined by direct cycle sequencing. The alignment of the deduced amino acid sequence derived from the PCR product and the m5C-MTase protein of FV3 revealed a homology of 55.4% identity and 29.1% similarity. The amino acid sequence which was found to be significantly homologous to the amino acid sequence deduced from the nucleotide sequence of the PCR product was located at the amino acid position 37 to 175 of the m5C-MTase of FV3 indicating the specificity of the amplified PCR product. The DNA nucleotide sequence of the LCDV-1 genome corresponding to the 5' and 3' termini of the m5C-MTase gene was determined by primer walking. The locus of the m5C-MTase gene of LCDV-1 was identified within the EcoRI DNA fragment G of LCDV-1 (7.9 kbp; 0.947 to 0.034 map units). The m5C-MTase gene of LCDV-1 comprises 684 nucleotides coding for a putative protein of 228 amino acid residues. A high degree of amino acid sequence homology (53.3% identity and 25.8% similarity) was detected between the m5C-MTase of LCDV-1 and FV3.

Amino Acid Sequence↗
PubMed1982

Mechanism of pBR322 transduction mediated by cytosine-substituting T4 bacteriophage.

A cytosine-substitution type mutant of bacteriophage T4 (T4dC phage) has been shown to mediate the transfer of plasmid pBR322. The transduction frequency was around 10(-2) per singly infected cell at low multiplicity of infection. The transductants contained either a monomer or multimers of pBR322. The transducing capacity of T4dC phage was resistant to methylmethanesulfonate treatment. The results of Southern blotting experiments have indicated that the pBR322 DNA exists as head-to-tail concatemers in the transducing particles. The mechanism of transfer of pBR322 mediated by T4dC phages is discussed.

Cytosine↗
PubMed1985

Different roles for cytosine methylation in HLA class II gene expression.

We studied the role of cytosine methylation in the control of HLA class II gene expression in isogenic sets of cells whose members differ in their expression of HLA class II genes. These included: T5-1, 6.1.6, and P30, which are a class II expressing B-cell line, a class II nonexpressing mutant derived from T5-1, and an HLA-DR expressing partial revertant derived from 6.1.6, respectively; the class II expressing B-cell line, SB, and the class II non-expressing T-cell line, HSB, from the same individual. The use of sets of cells that differ in the way their class II genes are regulated allows us to study how that difference is reflected in the methylation state of their class II genes. At least five out of six class II genes in nonexpressing cells have a CpG site that is demethylated, when compared with the same class II gene in the respective expressing cells. The results presented in this paper indicate that most methylation changes in and around class II genes have a correlation with their state of expression. Some of these changes reflect rather than determine the state of expression. Other methylation changes appear to directly affect expression, whereas some methylation differences neither correlate with nor influence gene expression. Although 5-azacytidine does not affect class II expression in T5-1 or 6.1.6, it does induce expression in HSB. This indicates that the basis for nonexpression of class II genes is different in 6.1.6 and HSB.

B-Lymphocytes↗
PubMed1986

DNA cytosine methylation and heat-induced deamination.

The heat-induced conversion of 5-methylcytosine (m5C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37 degrees C and pH 7.4 were approximately 9.5 X 10(-10) and 2.1 X 10(-10) sec-1, respectively. N4-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N4-methylcytosine rather than m5C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m5C in their DNA.

5-Methylcytosine↗
PubMed1984

5'-Methyl-cytosine in the macronuclear DNA of Blepharisma japonicum.

Brief report on the presence of 5'-methyl-cytosine as a minor base (0.56%) in the macronuclear DNA of the ciliate protozoan Blepharisma japonicum. The evidence comes from electrophoresis of macronuclear DNA digested by appropriate restriction endonucleases and high-performance liquid chromatography.

5-Methylcytosine↗
PubMed1988

The effects of guanine and cytosine variation on dinucleotide frequency and amino acid composition in the human genome.

One hundred twelve human DNA sequences were analyzed with respect to dinucleotide frequency and amino acid composition. The variation in guanine and cytosine (G + C) content revealed: (1) at 2-3 and 3-1 doublet positions CG discrimination is attenuated at high G + C, but TA disfavor is enhanced, and (2) several amino acids are subject to G + C change. These findings have been reported in part for collections of sequences from various species. The present study confirms that in a single organism--the human--the G + C effects do exist. Aspects of the argument that connects G + C with protein thermal stability are also discussed.

Animals↗
PubMed2004

Recombinant adenoviral vector containing tumor-specific L-plastin promoter fused to cytosine deaminase gene as a transcription unit: generation and functional test.

The expression of therapeutic transgenes in recombinant adenoviral vectors is a major cause of toxicity in dividing cancer cells as well as non dividing normal cells. To solve the problem of toxicity to normal cells, we have reported on a recombinant adenoviral vector system (AdLP-) in which the expression of the transgene is directed by the tumor-specific L-plastin promoter (LP) (Chung et al., 1999). The object of this study was to generate a recombinant adenoviral vector system which would generate tumor cell specific expression of cytosine deaminase (CD) gene. We report the construction of a replication-incompetent adenoviral vector in which CD is driven by the L-plastin promoter (AdLPCD). Infection of 293 cells by AdLPCD generated the functional CD protein as measured by HPLC analysis for the conversion of 5-Fluorocytosine (5-FC) to 5-Fluorouracil (5-FU). HPLC analysis in conjunction with counting radioactivity for [6-3H]-5FC and [6-3H]-5FU demonstrated vector dose-dependent conversion of 5-FC to 5-FU in AdLPCD infected ovarian cancer cells. The results from present and previous studies (Peng et al., 2001; Akbulut et al., 2003) suggest that the use of the AdLPCD/5-FC system may be of value in the treatment of cancer including microscopic ovarian cancer in the peritoneal cavity.

Adenoviridae↗
PubMed2000

Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene, single-copy transgenic locus in rice.

We investigated transgene silencing in a line of rice plants that carries a single-copy 6.6-kb transgenic locus comprising three heterologous transgenes: bar, hpt and gusA. We identified at least three distinct types of silencing effects associated with different methylation patterns, including a novel form of transcriptional silencing involving methylation of cytosine residues only at non-conventional acceptor sites in the coding region. Silencing arose de novo in individual R1, R2 and R3 plants despite the stability of the transgenic locus, although the basic structure of the locus, transgene dosage and position effects remained constant within the line. We found that different silencing effects could occur concurrently in adjacent heterologous transgenes in the same plant, with no evidence for spreading of silenced states or methylation patterns from one transgene to another.

Base Sequence↗
PubMed2004

Epigenetic tinkering and evolution: is there any continuity in the role of cytosine methylation from invertebrates to vertebrates?

The function of DNA methylation has been investigated in depth in vertebrate and plant genomes, establishing that it is involved in gene silencing and transposon control. Data regarding insect methylation, even if still scanty, apparently argue against evolutionary conservation of DNA methylation functions. Cytosine methylation, therefore, proves to be an epigenetic tool repeatedly used to accomplish different functions in different taxa according to a sort of epigenetic tinkering occurring during evolution.

Animals↗
PubMed2002

Aerobiosis increases the genomic guanine plus cytosine content (GC%) in prokaryotes.

The huge variation in the genomic guanine plus cytosine content (GC%) among prokaryotes has been explained by two mutually exclusive hypotheses, namely, selectionist and neutralist. The former proposals have in common the assumption that this feature is a form of adaptation to some ecological or physiological condition. On the other hand, the neutralist interpretation states that the variations are due only to different mutational biases. Since all of the traits that have been proposed by the selectionists either appeared to be limited to certain genera or were invalidated by the availability of more data, they cannot be considered as a selective force influencing the genomic GC% across all prokaryotes. In this report we show that aerobic prokaryotes display a significant increment in genomic GC% in relation to anaerobic ones. This is the first time that a link between a metabolic character and GC% has been found, independently of phylogenetic relationships and with a statistically significant amount of data.

Aerobiosis↗
PubMed2003

Complementary antineoplastic activity of the cytosine nucleoside analogues troxacitabine (Troxatyl) and cytarabine in human leukemia cells.

PURPOSE: Troxacitabine (BCH-4556, l-(-)-OddC, Troxatyl) is a novel beta- l-nucleoside analogue with potent antineoplastic activity both in vitro and in several tumor models in vivo, and is presently in phase II clinical trials. The combination of the cytosine analogues troxacitabine and araC (1-beta- d-arabinofuranosylcytosine, cytarabine) has shown promising activity in patients with acute myelogenous leukemia. To further examine the interactions between these two analogues, we investigated the in vitro and in vivo effects of their combination against a human leukemia cell line, CCRF-CEM. METHODS: . The in vitro cytotoxic effect of the combination of troxacitabine and araC on the survival of CCRF-CEM cells was measured using a standard MTT assay and combination indices were generated with the CalcuSyn software. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on survival of CCRF-CEM tumor-bearing animals. Mechanistic studies addressed recovery of DNA synthesis, intracellular levels of araC metabolites, feedback inhibition by triphosphate species and pharmacokinetics of both drugs. RESULTS: The combination of troxacitabine and araC in vitro was synergistic with combination indices between 0.1 and 0.7. This appeared to be related to the impact of the combination on DNA synthesis recovery, which was significantly delayed following exposure to the combination of troxacitabine and araC compared to either agent alone. Analysis of the effect of troxacitabine on the intracellular metabolites of araC revealed that troxacitabine did not inhibit araC deamination and caused a slight decrease in the overall intracellular accumulation of araCTP. The lower accumulation of araCTP could not be attributed to feedback inhibition caused by troxacitabine triphosphate on dCK. Furthermore, our in vivo experiments demonstrated that the combination of araC and troxacitabine was better at slowing down the progression of leukemia in SCID mice than either agent used alone without additive toxicities. Injections of 10 mg/kg troxacitabine i.p. daily for 5 days in combination with araC at 10 mg/kg led to an increase in median survival time of 58 days compared to 49.5 and 53.5 days for araC and troxacitabine, respectively, given as single agents. This represents an increase in life span of 17%, respectively when compared to araC alone. A pharmacokinetic study revealed that troxacitabine did not influence the disposition of araC when coadministered. CONCLUSIONS: Overall, our results show that the antileukemic activity of troxacitabine and araC is complementary when the two nucleoside analogues are combined in vivo. These effects appear to be related to their interaction at the level of DNA repair rather than to pharmacokinetic interactions. These results encourage the use of troxacitabine and araC in combination in patients with acute leukemia.

Animals↗
PubMed1997

A genetic and functional analysis of the unusually large variable region in the M.AluI DNA-(cytosine C5)-methyltransferase.

The M.AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5'-AGCT-3'. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more "target-recognizing domains" (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M.AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M.AluI is in fact monospecific it was found that AGCT methylation represents only 80-90% of the methylating activity of this enzyme, while control experiments with the enzyme M.HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M.AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M.AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M.AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.

Amino Acid Sequence↗
PubMed1999

Metal ions in non-complementary DNA base pairs: an ab initio study of Cu(I), Ag(I), and Au(I) complexes with the cytosine-adenine base pair.

Ab initio calculations have been carried out to characterize the structure and energetics of a silver(I) complex with the cytosine-adenine DNA base pair and an aqua ligand in the coordination sphere of Ag. In addition, we have also studied analogous complexes with Cu(I) and Au(I), and structures in which adenine has been replaced by purine in order to investigate the structural role of the adenine amino group. The calculations revealed that all metal-modified structures are dominated by the metal-base interactions, while the water-metal ion interaction and many-body interligand repulsion are less important contributions. Nevertheless, the structural role of the water molecule in the complex is quite apparent and in agreement with an earlier crystallographic study. The metal-modified base pairs exhibit large conformational flexibility toward out-of-plane motions (propeller twist and buckle), comparable or, in some cases, even larger than that observed in the base pairs without metal ions. All structures have been optimized within the Hartree-Fock approximation, while interaction energies were evaluated with the inclusion of electron correlation.

Adenine↗
PubMed2006

Pt-bridges in various single-strand and double-helix DNA sequences. DFT and MP2 study of the cisplatin coordination with guanine, adenine, and cytosine.

In this study, various platinum cross-links in DNA bases were explored. Some of these structures occur in many cis/trans-platinated double-helixes or single-stranded adducts. However, in the models studied, no steric hindrance from sugar-phosphate backbone or other surroundings is considered. Such restrictions can change the bonding picture partially but hopefully the basic energy characteristics will not be changed substantially. The optimization of the structures explored was performed at the DFT level with the B3LYP functional and the 6-31G(d) basis set. Perturbation theory at the MP2/6-31++G(2df,2pd) level was used for the single-point energy and 6-31+G(d) basis set for the electron-property analyses. It was found that the most stable structures are the diguanine complexes followed by guanine-cytosine Pt-cross-links, ca 5 kcal mol(-1) less stable. The adenine-containing complexes are about 15 kcal mol(-1) below the stability of diguanine structures. This stability order was also confirmed by the BE of Pt-N bonds. For a detailed view on dative and electrostatic contributions to Pt-N bonds, Natural Population Analysis, determination of electrostatic potentials, and canonical Molecular Orbitals description of the examined systems were used.

Adenine↗
PubMed1987

Spectropolarimetric determination of the guanine-cytosine content of DNA.

A circular dichroism technique for the determination of DNA base composition in terms of the guanine-cytosine content has been developed. A linear relationship between the ratio of ellipticities at 260 and 280 nm (theta 260/theta 280) and base composition of DNA is obtained in citrate-phosphate buffer, pH 3.0 at 20 degrees C. The DNA base composition obtained with this method is not only highly reproducible but is also the fastest method reported.

Base Composition↗