Remarkable enhancement in the DNA-binding ability of C2-fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines and their anticancer potential

C2-Fluoro substituted DC-81, and its dimers that comprise of two C2-fluoro substituted DC-81 subunits tethered to their C8-position through simple alkane spacers as well as piperazine moiety side-armed with symmetrical alkyloxy spacers have been designed and synthesized. These fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines have shown remarkable DNA-binding ability and most of them possess promising anticancer activity, having GI₅₀ values in micromolar to nanomolar concentration range. DNA thermal denaturation studies show that some of these compounds (14a–c and 15) increase the ΔT_m values in the range of 28.9–38 °C, and this is further confirmed by the restriction endonuclease studies. This study illustrates the importance of introducing fluoro substitution at the C2-position apart from the incorporation of a piperazine ring in between the alkyloxy linker for enhancement of the DNA-binding ability in comparison to DSB-120 and SJG-136 (ΔT_m = 10.2 and 25.7 °C). Moreover, the variations in the DNA-binding ability with respect to fluoro substitution in this class of dimers has been investigated by molecular modeling studies. Some representative C2-fluoro substituted dimers (8a and 14a) have also exhibited significant anticancer activity in the 60 cancer cell line assay of the National Cancer Institute (NCI).     

Relationship between cytotoxicity and DNA-binding affinity of amidine derivatives of tetrahydroquino[4,3-b][1]benzazepines and tetrahydrobenzo[k]naphthyridines

Two sets of amidine derivatives of the non-planar tetracyclic systems: tetrahydroquino[4.3-b][1]benzazepine (I) and tetrahydrobenzo[k]naphthyridine (II), bearing three types of side chains (hydroxyl, amine and alkyl) have been synthesized. All the compounds were found to possess weak but significant DNA-binding affinity which correlated with in vitro cytotoxicity across two cell lines.     

Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix?. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix? vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.     

Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.     

3H]U69,593 binding in guinea-pig brain: comparison with [3H]ethylketazocine binding at the kappa-opioid sites

[3H]U69,593 and [3H]ethylketazocine (mu + delta suppressed) binding was measured in homogenates of guinea-pig brain. Both ligands bind with high affinity to a single class of opioid sites. The relative equilibrium dissociation constant (KD) for [3H]U69,593 is 1.15 nM, while [3H]ethylketazocine has a KD value of 0.33 nM. Their respective maximum binding capacities are 4.49 and 4.48 pmol/g of wet tissue. Various mu-selective, delta-selective, kappa-selective, and nonselective opioids were tested in competition studies against the binding of [3H]U69,593 or [3H]ethylketazocine (in the presence of mu- and delta-blockers) to measure their relative affinity. [D-Ala2, MePhe4,Gly5-ol]enkephalin (mu-selective) has low affinity (600-3000 nM) and [D-Pen2,D-Pen5]enkephalin and [D-Ser2, Leu5, Thr6]enkephalin (delta-selective) have very low affinities (greater than 20,000 nM) at the sites labelled with [3H]U69,593 or [3H]ethylketazocine. On the other hand, unlabelled U69,593, U50,488H, and tifluadom (all three kappa-selective substances) display high affinity (1-5 nM) at those sites. Nonselective opioids, such as bremazocine, levorphanol, and ethylketazocine show similar affinities at the sites labelled with [3H]U69,593 and at the sites labelled with [3H]ethylketazocine. These data indicate that [3H]U69,593 is a selective high-affinity ligand for the same sites that are labelled with [3H]ethylketazocine (in the presence of mu- and delta-blockers) and that these are kappa-sites.     

Reactivity of nitric oxide with the [4Fe-4S] cluster of dihydroxyacid dehydratase from Escherichia coli

Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.     

Synthesis, characterization and DNA-binding of novel chiral complexes delta- and lambda-[Ru(bpy)2L]2+ (L = o-mopip and p-mopip)

Novel chiral Ru(II) complexes [Ru(bpy)2L]2+ (bpy = 2,2-bipyridine; L: o-mopip = 2-(2-methoxylphenyl)imidazo[4,5-f][1,10]phenanthroline, p-mopip = 2-(4-methoxylphenyl)imidazo[4,5-f][1,10]phenanthroline) containing -OCH3 at different positions on the phenyl ring have been synthesized and characterized. The DNA-binding and DNA-photocleavage properties of the complexes were investigated. The theoretical calculations for these complexes were also carried out applying the density functional theory (DFT) method. The experimental results show that: both these two isomer complexes can bind to DNA in an intercalative mode; the DNA-binding affinity of [Ru(bpy)2(p-mopip)] 2 is greater than that of [Ru(bpy)2(o-mopip)] 1; moreover, the DNA-binding affinities of enantiomers delta-1 and delta-2 are all greater than those of lambda-1 and lambda-2, respectively. In addition, a very interesting finding is experimentally obtained, i.e. under a low [DNA]/[Ru] ratio, the emission intensities of delta-1 and lambda-1 are all weaker than those of delta-2 and lambda-2, however, upon a high [DNA]/[Ru] ratio, the emission intensities of both delta-1 and lambda-1 are stronger than those of delta-2 and lambda-2. Such a difference of the emission spectra can be interpreted by the electric effect of substituent on the intercalative ligand. The difference in DNA-binding affinities of these two isomeric complexes can also be reasonably explained by the DFT calculations.     

Reversible inactivation of E. coli endonuclease III via modification of its [4Fe-4S] cluster by nitric oxide

Endonuclease III, a highly conserved enzyme initiating the base excision repair of oxidized DNA bases, hosts a [4Fe-4S] cluster. Unlike many other iron-sulfur clusters, the [4Fe-4S] cluster of endonuclease III is stable and resistant to both oxidation and reduction. Here we show that the [4Fe-4S] cluster of the E. coli endonuclease III can be readily modified by nitric oxide forming the protein-bound dinitrosyl iron complex in vitro and in vivo. Modification of the [4Fe-4S] cluster completely inhibits the DNA glycosylase activity of the endonuclease III. Remarkably, the enzymatic activity is restored when the [4Fe-4S] cluster is re-assembled in the endonuclease III dinitrosyl iron complex with L-cysteine, cysteine desulfurase (IscS) and ferrous iron in vitro. Furthermore, the nitric oxide-modified [4Fe-4S] cluster in endonuclease III is efficiently repaired in aerobically growing E. coli cells, and this repair does not require new protein synthesis. These results suggest that the E. coli endonuclease III can be reversibly inactivated by nitric oxide via modification of its [4Fe-4S] cluster.     

Syntheses and DNA-binding studies of two ruthenium(II) complexes containing one ancillary ligand of bpy or phen: [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)2

Two new ruthenium(II) complexes of [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)(2) (bpy=2,2'-bipyridine, phen=1,10-phenanthroline, pp[2,3]p=pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) have been synthesized and characterized by elemental analysis and 1H NMR spectra. The calf thymus DNA-binding properties of the two complexes were investigated by UV-visible and emission spectroscopy, competitive binding experiments with ethidium bromide and viscosity measurements. The results indicate that the two complexes intercalate between the base pairs of the DNA tightly with intrinsic DNA-binding constants of 3.08 x 10(6) and 6.53 x 10(6) M(-1) in buffered 50 mM NaCl, respectively, which are much larger than 6.9 x 10(5) M(-1) for [Ru(bpy)2(pp[2,3]p)](ClO4)2 containing two ancillary ligands of bpy.     

DNA binding and cleavage selectivity of the Escherichia coli DNA G:T-mismatch endonuclease (vsr protein).

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The gene encoding the vsr endonuclease is next to the gene specifying the E. coli dcm DNA-methyltransferase; an enzyme that adds CH3 groups to the first dC within its target sequence CC[A/T]GG, giving C5MeC[A/T]GG. Deamination of the d5MeC results in CT[A/T]GG in which the first T is mis-paired with dG and it is believed that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bases surrounding the T:G mismatch has been evaluated. Determination of specificity constant (kst/KD; kst = rate constant for single turnover, KD = equilibrium dissociation constant) confirms vsr's preference for a T:G mismatch within a dcm sequence i.e. CT[A/T]GG (the underlined T being mis-paired with dG) is the best substrate. However, the enzyme is capable of binding and hydrolysing sequences that differ from the dcm target site by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease has a much lower selectivity for the dcm sequence than type II restriction endonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible physiological role.     

Identification of preferred actinomycin-DNA binding sites by the combinatorial method REPSA.

An important question in the study of ligand-DNA interactions is the determination of binding specificity. Here, we used the combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) to identify the preferred duplex DNA-binding sites of the antineoplastic agent actinomycin D. After 10 rounds of REPSA, over 95% of the cloned DNAs exhibited significantly reduced FokI restriction endonuclease cleavage in the presence of 1 microM actinomycin. A chi(2) statistical analysis of their sequences found that 39 of the 45 clones contained one or more copies of the sequence 5'-(T/A)GC(A/T)-3', giving a p<0.001 for this consensus. A DNase I footprinting analysis of the cloned DNAs found that all possessed relatively high affinity actinomycin-binding sites with apparent dissociation constants between 12 and 258nM (average 98nM). The average footprint encompassed 7.6 bases and in most cases (90%) included one or more consensus sequences. Interestingly, several of the selected clones contained overlapping consensus sequences (e.g., 5'-TGCTGCT-3'), suggesting that such close proximity DNA-binding sites may actually be preferred by actinomycin under physiological conditions.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Role of steric interactions in the delta opioid receptor selectivity of [D-Pen2, D-Pen5]enkephalin

In order to assess the individual effects of each of the 3-methyl groups in residue 2 of [D-Pen2, D-Pen5]enkephalin on binding affinity to mu and delta opioid receptors, (2S,3S)methylcysteine ((3S)Me-D-Cys) and (2S,3R)methylcysteine ((3R)Me-D-Cys) were synthesized and incorporated into the analogs, [(3S)Me-D-Cys2, D-Pen5] enkephalin and [(3R)Me-D-Cys2, D-Pen5]enkephalin. Of these analogs, [(3S)Me-D-Cys2, D-Pen5]enkephalin appears from 1H n.m.r. spectra to assume a conformation similar to those of [D-Pen2, D-Pen5]enkephalin and the less delta receptor-selective, but more potent, [D-Cys2, D-Pen5]enkephalin. Assessment of binding affinity to mu and delta receptors revealed that [(3S)Me-D-Cys2, D-Pen5]enkephalin exhibits delta receptor affinity intermediate between [D-Pen2, D-Pen5]enkephalin and [D-Cys2, D-Pen5]enkephalin while its mu receptor affinity is similar to that of [D-Cys2, D-Pen5]enkephalin. These results suggest that, for [D-Pen2, D-Pen5]enkephalin, adverse steric interactions between the D-Pen2 pro-R methyl group and the mu receptor binding site lead to the low mu receptor binding affinity observed for this analog. By contrast, both the pro-R and pro-S D-Pen2 methyl groups lead to minor steric interactions which contribute to the somewhat lower delta receptor affinity of this compound.     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

Heterogeneity of human lipoprotein Lp[a]: cytochemical and biochemical studies on the interaction of two Lp[a] species with the LDL receptor

Human Lp[a] can be fractionated into two species with different affinities for lysine-Sepharose. Forty to 81% of the total Lp[a] in the density fraction 1.055-1.15 g/ml from five individuals was retained by this affinity column. The remaining unretained Lp[a] species with no apparently functional lysine binding site was similar to the retained species in its electrophoretic mobility, lipid, protein, and apolipoprotein composition, and the heterogeneity was not related to apo[a] size polymorphism. Interaction of the two species with the low density lipoprotein (LDL) receptor was studied in human fibroblasts. Using gold-labeled lipoproteins and an immunochemical procedure, both Lp[a] species could be located in clusters on the cell surface, but the extent of labeling was far lower than that seen with LDL. Both Lp[a] variants were less effective than LDL in 1) down-regulation of LDL-receptor activity; 2) suppression of cellular sterol synthesis; and 3) stimulation of cholesteryl ester formation in human fibroblasts. Although degradation of both species of Lp[a] by the perfused rat liver was stimulated after estrogen induction of hepatic LDL-receptor activity, the stimulation amounted to only a quarter of that seen with LDL. The heterogeneity of Lp[a] with respect to the ability to bind epsilon-aminocarboxylic acid will need to be considered in studying the physiological role of this lipoprotein. Both Lp[a] species exhibited a similar interaction with the LDL-receptor in vitro, and confirmed previous investigations that Lp[a] is only a poor ligand for the LDL-receptor.     

Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs.

BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector.     

Synthesis and biological evaluation of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) C8 cyclic amine conjugates

We report examples of a series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) analogues 12-15 prepared from a common functionalized building block 11 that can be conveniently synthesized on a large scale and in optically pure form. Isoindoline analogue 15 is the most cytotoxic agent in this series, has the highest DNA-binding affinity, and shows significant activity in the in vivo hollow fibre assay.     

Synthesis of the bicyclo-[4.3.0]-thymidyl-nucleoside via pd(II)-mediated ring expansion chemistry

A variety of modified nucleosides to improve antisense oligodeoxynucleotide properties such as target affinity, nuclease resistance, and pharmacokinetics were developed in the last two decades. In the context of conformational restriction we present here the synthesis of the [4.3.0]-bicyclo-DNA thymine monomer via Pd(II)-mediated ring expansion of an intermediate of the tricyclo-DNA synthesis.     

2,7-diazabicyclo[3.3.0]octanes as novel h5-HT receptor agonists.

The conformational restriction of a (benzylamino)methyl substituted pyrrolidine to form 2,7-diazabicyclo[3.3.0]octanes has led to a series of compounds with high affinity at the h5-HT1D receptor as well as dramatically increased concentrations in the hepatic portal vein following oral administration.     

Cloning and sequencing of LlaDCHI [corrected] restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system.

The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp. cremoris DCH-4. It encodes a restriction/modification system named LlaDCHI [corrected]. When introduced into a phage-sensitive L. lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335. The LlaDCHI [corrected] endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3'. It is an isoschizomer of Streptococcus pneumoniae DpnII. The plasmid pSRQ700 was mapped, and the genetic organization of LlaDCHI [corrected] was localized. Cloning and sequencing of the entire LlaDCHI [corrected] system allowed the identification of three open reading frames. The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter. A putative terminator was found at the end of llaIIC. The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease. The LlaDCHI [corrected] system shares strong genetic similarities with the DpnII system. The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA. However, R.LlalII shared only 31% identity with R.DpnII.