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Biomedical subjects

A Janulaitis

Publications and source records attributed to A Janulaitis.

52 records · Page 3Linked to original sources
PubMed1989

Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases.

The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.

Amino Acid Sequence↗
PubMed1987

Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine.

The synthesis of N4-methyl-2'-deoxycytidine and its fully protected mononucleotide, suitable for the oligonucleotide synthesis by phosphotriester method is described. A set of octanucleotides - d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG) and dodecanucleotides - d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), d(GGA4mCCCGGGTCC) has been synthesized in a solution. Physical characterization of the oligonucleotide duplexes by means of UV and CD spectrometry provides the evidence that 4mC similarly to 5mC favours the B--greater than Z transition, although both of these methylated cytosines inhibit the B--greater than A conformational change. N4-Methylcytosine in contrast to 5-methylcytosine reduces the DNA double helix thermal stability.

5-Methylcytosine↗
PubMed1987

Cleavage of methylated CCCGGG sequences containing either N4-methylcytosine or 5-methylcytosine with MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases.

The cleavage specificity of R.Cfr9I was determined to be C decreases CCGGG whereas the methylation specificity of M.Cfr9I was C4mCCGGG. The action of MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases on an unmethylated parent d(GGACCCGGGTCC) dodecanucleotide duplex and a set of oligonucleotide duplexes, containing all possible substitutions of either 4mC or 5mC for C in the CCCGGG sequence, was investigated. It was found that 4mC methylation, in contrast to 5mC, renders the CCCGGG site resistant to practically all the investigated endonucleases. The cleavage of methylated substrates with restriction endonucleases is discussed.

5-Methylcytosine↗
PubMed1987

Interaction of AluI, Cfr6I and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine.

The cleavage specificity of R.Cfr6I, an isoschizomer of PvuII restriction endonuclease was determined to be 5'CAG decreases CTG and the methylation specificity of Cfr6I and PvuII methylases, 5'CAG4mCTG. Thus, M.Cfr6I and M.PvuII are new additions to the list of methylases with N4-methylcytosine specificity. Neither of the above RM enzymes acts on the substrates containing either N4-methylcytosine or 5-methylcytosine in a cognate methylation position.

5-Methylcytosine↗
PubMed1985

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence.

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of lambda DNA have been compared. A common 5'GAGACC 3'CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5' GGTCTCN decreases 3' CCAGAGN NNNN increases.

Base Sequence↗
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↗
PubMed1983

A new site-specific endodeoxyribonuclease from Citrobacter freundii.

Cfr10 I, a site-specific endonuclease from Citrobacter freundii strain RFL10, was isolated. It recognizes and cleaves the family of related sequences: 5'Pu decreases CCGGPy to generate DNA fragments with 5' tetranucleotide extensions. Cfr10 I may be useful in molecular cloning experiments, especially in conjunction with other enzymes which generate the same terminal extensions.

Base Sequence↗
PubMed1983

Three sequence-specific endonucleases from Escherichia coli RFL47.

The characterization of the new restriction enzyme Eco47III recognizing a hexanucleotide palindromic sequence 5'AGC decreases GCT and cleaving, as indicated by the arrow, is reported. It was isolated from Escherichia coli strain RFL47. Another two specific endonuclease Eco47I (isoschizomer of AvaII) and Eco47II (isoschizomer of AsuI) were also found in this strain. There are two Eco47III recognition sites on lambda DNA at 20997 and 37060 basepairs. The central Eco47III fragment can be replaced by a cloned fragment in lambda vector mutant in tR2 gene; i.e., lambda gt.

Base Sequence↗
PubMed1982

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli.

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.

Bacillus↗
PubMed1982

Restriction map of the hepatitis B virus DNA cloned in Escherichia coli.

A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases allows to conclude that the cloned DNA is similar, but not identical to the HBV DNA of ayw subtype.

Chromosome Mapping↗
PubMed1982

Isolation and some properties of the restriction endonuclease BcnI from Bacillus centrosporus.

A specific type-II restriction endonuclease BcnI from Bacillus centrosporus has been purified to electrophoretic homogeneity in three chromatographic steps. Around 15 micrograms of such a preparation can be isolated from 1 g of the cell paste. The yield of the enzyme is higher than that of any type-II restriction endonuclease so far reported. The molecular weight of the enzyme determined by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate equals 27 500 and 28 000 respectively. The activity of the restriction endonuclease is maximal at pH 9.2 and 40--45 degrees C. The optimal magnesium concentration was estimated to be 7.5 mM. The activity of BcnI may also be observed in the presence of Co2+, Mn2+, Ni2+ and Zn2+ but it is markedly less than in the presence of Mg2+.

Bacillus↗