scholarly journals M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1111-1121 ◽  
Author(s):  
Beata Furmanek-Blaszk ◽  
Robert Boratynski ◽  
Natalia Zolcinska ◽  
Marian Sektas
Keyword(s):  
Functional Analysis ◽  
Divalent Cations ◽  
Restriction Enzymes ◽  
Bacterial Cells ◽  
Recognition Sequence ◽  
Moraxella Bovis ◽  
Chromatographic Procedure ◽  
Methylation Activity ◽  
Restriction Modification ◽  
Endonuclease Gene

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction–modification (R–M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5′-GAAGA-3′/3′-CTTCT-5′. M1.MboII modifies the last adenine in the recognition sequence 5′-GAAGA-3′ to N 6-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3′-CTTCT-5′, yielding N 4-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000±1000 Da under denaturing conditions. Divalent cations (Ca2+, Mg2+, Mn2+ and Zn2+) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R–M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.

scholarly journals The Influence of 5′R and 5′S cdA and cdG on the Activity of BsmAI and SspI Restriction Enzymes

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3750
Author(s):  
Michał Szewczuk ◽  
Karolina Boguszewska ◽  
Julia Kaźmierczak-Barańska ◽  
Bolesław T. Karwowski
Keyword(s):  
Genetic Material ◽  
Restriction Enzymes ◽  
Dna Lesions ◽  
Recognition Site ◽  
Recognition Sequence ◽  
Cellular Mechanisms ◽  
Modification System ◽  
Restriction Modification System ◽  
Restriction Modification ◽  
Oligonucleotide Sequence

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5′,8-cyclo-2′-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction–modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme’s recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5′S)-5′,8-cyclo-2′-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5′R)-5′,8-cyclo-2′-deoxyadenosine (RcdA). Interestingly, in the case of 5′,8-cyclo-2′-deoxyguanosine (cdG), both 5′S and 5′R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.

scholarly journals Lactococcal Plasmid pNP40 Encodes a Novel, Temperature-Sensitive Restriction-Modification System

2004 ◽  
Vol 70 (9) ◽  
pp. 5546-5556 ◽  
Author(s):  
Jonathan O'Driscoll ◽  
Frances Glynn ◽  
Oonagh Cahalane ◽  
Mary O'Connell-Motherway ◽  
Gerald F. Fitzgerald ◽  
...  
Keyword(s):  
Restriction Enzymes ◽  
Temperature Sensitive ◽  
Temperature Dependent ◽  
Modification System ◽  
Restriction Modification System ◽  
Naturally Occurring ◽  
Low Copy Number ◽  
Encoding Genes ◽  
Restriction Modification ◽  
Specific Mrna

ABSTRACT A novel restriction-modification system, designated LlaJI, was identified on pNP40, a naturally occurring 65-kb plasmid from Lactococcus lactis. The system comprises four adjacent similarly oriented genes that are predicted to encode two m5C methylases and two restriction endonucleases. The LlaJI system, when cloned into a low-copy-number vector, was shown to confer resistance against representatives of the three most common lactococcal phage species. This phage resistance phenotype was found to be strongly temperature dependent, being most effective at 19°C. A functional analysis confirmed that the predicted methylase-encoding genes, llaJIM1 and llaJIM2, were both required to mediate complete methylation, while the assumed restriction enzymes, specified by llaJIR1 and llaJIR2, were both necessary for the complete restriction phenotype. A Northern blot analysis revealed that the four LlaJI genes are part of a 6-kb operon and that the relative abundance of the LlaJI-specific mRNA in the cells does not appear to contribute to the observed temperature-sensitive profile. This was substantiated by use of a LlaJI promoter-lacZ fusion, which further revealed that the LlaJI operon appears to be subject to transcriptional regulation by an as yet unidentified element(s) encoded by pNP40.

scholarly journals Expression and functional analysis of the nobiletin biosynthesis-related gene CitOMT in citrus fruit

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mao Seoka ◽  
Gang Ma ◽  
Lancui Zhang ◽  
Masaki Yahata ◽  
Kazuki Yamawaki ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Citrus Fruit ◽  
Satsuma Mandarin ◽  
Important Health ◽  
Nutritional Qualities ◽  
Citrus Varieties ◽  
Ponkan Mandarin ◽  
Methylation Activity ◽  
New Strategies

Abstract Nobiletin, a polymethoxy flavone (PMF), is specific to citrus and has been reported to exhibit important health-supporting properties. Nobiletin has six methoxy groups at the 3′,4′,5,6,7,8-positions, which are catalyzed by O-methyltransferases (OMTs). To date, researches on OMTs in citrus fruit are still limited. In the present study, a novel OMT gene (CitOMT) was isolated from two citrus varieties Satsuma mandarin (Citrus unshiu Marc.) and Ponkan mandarin (Citrus reticulata Blanco), and its function was characterized in vitro. The results showed that the expression of CitOMT in the flavedo of Ponkan mandarin was much higher than that of Satsuma mandarin during maturation, which was consistent with the higher accumulation of nobiletin in Ponkan mandarin. In addition, functional analysis showed that the recombinant protein of CitOMT had methylation activity to transfer a methyl group to 3′-hydroxy group of flavones in vitro. Because methylation at the 3′-position of flavones is vital for the nobiletin biosynthesis, CitOMT may be a key gene responsible for nobiletin biosynthesis in citrus fruit. The results presented in this study will provide new strategies to enhance nobiletin accumulation and improve the nutritional qualities of citrus fruit.

NUTRITION AND METABOLISM OF MARINE BACTERIA: XI. SOME CHARACTERISTICS OF THE LYTIC PHENOMENON

1962 ◽  
Vol 8 (6) ◽  
pp. 883-896 ◽  
Author(s):  
Robert A. MacLeod ◽  
Tibor I. Matula
Keyword(s):  
Cell Wall ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Morphological Characteristics ◽  
Bacterial Cells ◽  
Whole Cells ◽  
Lytic Action ◽  
Absorbing Materials ◽  
Sulphate Salts ◽  
Osmotic Effects

Five marine bacteria examined were found to differ considerably in lytic susceptibility. Some lysed completely below 0.15 M NaCl while suspensions of others contained some whole cells at 0.025 M NaCl. In general NaCl and LiCl were more effective than KCl or NH4Cl in protecting against lysis and the loss of ultraviolet-absorbing materials from the cells. KCl could spare the requirement for NaCl to prevent lysis. Mg++ and other divalent cations at 0.05 M or less completely prevented lysis of all but one of the organisms. Sulphate salts stabilized the cell suspensions better on incubation than did chlorides for four of the organisms. For the fifth the reverse was true. The lytic action of ethanol and glycerol could be prevented by NaCl or sucrose. The concentrations of the latter required to prevent lysis remained unchanged when ethanol or glycerol was included in the suspending medium. Cells washed essentially free of electrolytes with 0.5 M sucrose retained their morphological characteristics. The observations are considered in relation to the distinction between marine bacteria, halophiles, and terrestrial bacteria and to current theories of the mechanism of lysis of bacterial cells. The results are not consistent with the hypotheses either that primarily osmotic effects are involved or that electrolytes are required to maintain the cell wall.

Binding of MmeI Restriction–modification Enzyme to its Specific Recognition Sequence is Stimulated by S-adenosyl-l-methionine

2007 ◽  
Vol 37 (2) ◽  
pp. 127-135 ◽  
Author(s):  
Joanna Nakonieczna ◽  
Jaroslaw W. Zmijewski ◽  
Bogdan Banecki ◽  
Anna J. Podhajska
Keyword(s):  
Recognition Sequence ◽  
Specific Recognition ◽  
Restriction Modification ◽  
Modification Enzyme

scholarly journals Four New Genes of Cyanobacterium Synechococcus elongatus PCC 7942 Are Responsible for Sensitivity to 2-Nonanone

2020 ◽  
Vol 8 (8) ◽  
pp. 1234
Author(s):  
Olga A. Koksharova ◽  
Alexandra A. Popova ◽  
Vladimir A. Plyuta ◽  
Inessa A. Khmel
Keyword(s):  
Cell Wall ◽  
Transport System ◽  
Molecular Mechanisms ◽  
Synechococcus Elongatus ◽  
Bacterial Cells ◽  
New Genes ◽  
Abc Transport ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942 ◽  
Restriction Modification

Microbial volatile organic compounds (VOCs) are cell metabolites that affect many physiological functions of prokaryotic and eukaryotic organisms. Earlier we have demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. Cyanobacteria are very sensitive to ketone action. To investigate the possible molecular mechanisms of the ketone 2-nonanone influence on cyanobacterium Synechococcus elongatus PCC 7942, we applied a genetic approach. After Tn5-692 transposon mutagenesis, several 2-nonanone resistant mutants have been selected. Four different mutant strains were used for identification of the impaired genes (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732, Synpcc7942_0726) that encode correspondingly: 1) a murein-peptide ligase Mpl that is involved in the biogenesis of cyanobacteria cell wall; 2) a putative ABC transport system substrate-binding proteins MlaD, which participates in ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane by aberrantly localized phospholipids transport from outer to inner membranes of bacterial cells; 3) a conserved hypothetical protein that is encoding by gene belonging to phage gene cluster in Synechococcus elongatus PCC 7942 genome; 4) a protein containing the VRR-NUC (virus-type replication-repair nuclease) domain present in restriction-modification enzymes involved in replication and DNA repair. The obtained results demonstrated that 2-nonanone may have different targets in Synechococcus elongatus PCC 7942 cells. Among them are proteins involved in the biogenesis and functioning of the cyanobacteria cell wall (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732) and protein participating in stress response at DNA restriction-modification level (Synpcc7942_0726). This paper is the first report about the genes that encode protein products, which can be affected by 2-nonanone.

scholarly journals suGF1 binds in the major groove of its oligo(dG).oligo(dC) recognition sequence and is excluded by a positioned nucleosome core.

1994 ◽  
Vol 14 (2) ◽  
pp. 1410-1418 ◽  
Author(s):  
D Patterton ◽  
J Hapgood
Keyword(s):  
Sea Urchin ◽  
Intergenic Region ◽  
Divalent Cations ◽  
Major Groove ◽  
Recognition Sequence ◽  
Nucleosome Core ◽  
Histone Octamer ◽  
Developmental Gene Regulation ◽  
Dna Complex

We have elsewhere reported the purification of a poly(dG).poly(dC)-binding nuclear protein (suGF1) from sea urchin embryos (J. Hapgood and D. Patterton, Mol. Cell. Biol. 14:this issue, 1994). We proposed that suGF1 may be a member of a family of G-string factors involved in developmental gene regulation, possibly via alterations in chromatin structure. In this article, we characterize the binding of purified suGF1 to 11 contiguous Gs in the H1-H4 intergenic region of a sea urchin early histone gene battery in vitro. It is shown that suGF1-DNA binding is dependent on ionic strength and requires divalent cations. Purified suGF1 forms discrete protein-DNA multimers, consistent with suGF1-suGF1 interactions. In a model for the suGF1-DNA complex derived from our footprinting and methylation interference data, suGF1 contacts the Gs in the major groove as well as one of the bordering phosphate backbones. The data are consistent with the direction of curvature of the DNA in the suGF1-DNA complex being the same as that preferred by the free DNA and exhibited by the DNA when bent around a positioned nucleosome core in vitro. However, on the basis of steric considerations, the binding of suGF1 and that of the histone octamer are predicted to be mutually exclusive. We show that suGF1 is indeed unable to bind to the G string when occupied by a histone octamer located in the major in vitro positioning frame in the H1-H4 intergenic region.

Banding of rye (Secale cereale) chromosomes using the restriction enzymes AluI, DraI, HpaII, and MspI

Genome ◽  
1993 ◽  
Vol 36 (5) ◽  
pp. 998-1002 ◽  
Author(s):  
T. Stößer ◽  
T. Günther ◽  
C. U. Hesemann
Keyword(s):  
Secale Cereale ◽  
Restriction Enzymes ◽  
Chromosome Band ◽  
Banding Technique ◽  
Recognition Sequence ◽  
Metaphase Chromosomes ◽  
Banding Patterns ◽  
Secale Cereale L ◽  
Characteristic Banding

Mitotic metaphase chromosomes of the rye inbred line L 301, which belongs to the Sortiment of the University of Hohenheim, were treated in situ with the restriction enzymes AluI (recognition sequence: 5′-AC/GT-3′), DraI (recognition sequence: 5′-TTT/AAA-3′), and the isoschizomeres HpaII and MspI (recognition sequence: 5′-C/CGG-3′) and stained with Giemsa. The chromosomes indicated similar banding patterns in comparison with the conventional Giemsa-C-banding. However, we have found in rye chromosomes after restrictase treatment that the telomeric bands were reduced in extension. In a lower degree the centromeric bands of individual chromosomes could be absent in dependence of the used restriction enzymes. The number of the intercalary bands were also reduced. Nevertheless, the tested restriction enzymes produced characteristic banding patterns of the rye genome. This uncomplicated banding technique is suited for a very quick banding method of karyotype analysis especially to obtain a first survey of the band patterns on the rye chromosomes.Key words: Secale cereale L., chromosome band pattern, in situ digestion, restriction endonuclease, restriction banding.

scholarly journals LraI fromLactococcus raffinolactisBGTRK10-1, an Isoschizomer ofEcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Marija Miljkovic ◽  
Milka Malesevic ◽  
Brankica Filipic ◽  
Goran Vukotic ◽  
Milan Kojic
Keyword(s):  
Restriction Enzyme ◽  
Specific Activity ◽  
Restriction Enzymes ◽  
Ion Concentration ◽  
Gene Encoding ◽  
Modification System ◽  
Star Activity ◽  
Restriction Modification System ◽  
Monovalent Ions ◽  
Restriction Modification

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity.Lactococcus raffinolactisBGTRK10-1 expressesLraI Type II restriction-modification enzyme, whose activity is similar to that shown forEcoRI;LraI methyltransferase protects DNA fromEcoRI cleavage. The gene encodingLraI endonuclease was cloned and overexpressed inE. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13°C and 37°C) and was stable after storage at −20°C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity ofLraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence forLraI restriction enzyme was determined as 5′-G/AATTC-3′, indicating thatLraI restriction enzyme is an isoschizomer ofEcoRI. In the reaction buffer with a lower salt concentration,LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5′-A/AATTC-3′, leaving the same sticky ends on fragments asEcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin ofLraI restriction-modification system with previously describedEcoRI-like restriction-modification systems.

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