Bacterial Restriction Endonuclease Research Articles

IDENTIFICATION OF LEPTOSPIRAL ISOLATES BY BACTERIAL RESTRICTION ENDONUCLEASE ANALYSIS (BRENDA)

DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.

Structure of BamHI Bound to Nonspecific DNA: A Model for DNA Sliding

The central problem faced by DNA binding proteins is how to select the correct DNA sequence from the sea of nonspecific sequences in a cell. The problem is particularly acute for bacterial restriction enzymes because cleavage at an incorrect DNA site could be lethal. To understand the basis of this selectivity, we report here the crystal structure of endonuclease BamHI bound to noncognate DNA. We show that, despite only a single base pair change in the recognition sequence, the enzyme adopts an open configuration that is on the pathway between free and specifically bound forms of the enzyme. Surprisingly, the DNA drops out of the binding cleft with a total loss of base-specific and backbone contacts. Taken together, the structure provides a remarkable snapshot of an enzyme poised for linear diffusion (rather than cleavage) along the DNA.

Understanding of molecular biology

To display theorical and methodological basis of the molecular biology. To point out its main medical applications. For this review, we analysed the English and French literature concerning the research and clinical aspects of the molecular biology, especially in anaesthesiology and intensive care, using the Medline database. The current textbooks were also used. We selected: 1) the original articles corresponding to the main advances that resulted in the present state of this discipline; 2) the reviews; 3) some chapters of textbooks. In this review, we report: 1) the current knowledge concerning the conservation and the expression of the genome; 2) the principles of the most widely used experimental techniques; 3) the medical applications of this knowledge in anaesthesiology and intensive care; 4) the more recent developments of this research field. Within medical biology, molecular biology essentially corresponds to the study of nucleic acids. In this review, the general principles governing the organization and expression of the genome are discussed. The expansion of molecular biology has been a consequence of the widespread use of enzymatic tools, of which bacterial restriction enzymes were the first. Numerous enzymes are now available, permitting DNA strands to be cut, linked, synthesized and sequenced. Several of the most representative molecular biology techniques are described. Some of them, such as PCR, are commonly used in clinical situations. Animal experimental models have also been generated by genome altering methods, in order to analyse the phenotypic consequences of these modifications. Recently, a viable mammal, deriving from a differentiated cell, has been cloned. Human embryonic totipotent stem cells are now available in cultures. These advances have important ethical implications whilst, at the same time, offering new opportunities for medical applications. The state of gene therapy and human genome sequencing programmes is discussed.

Cleavage of the HIV replication primer tRNALys,3 in human cells expressing bacterial anticodon nuclease

Anticodon nuclease is a bacterial restriction enzyme directed against tRNA(Lys). We report that anticodon nuclease also cleaves mammalian tRNA(Lys) molecules, with preference and site specificity shown towards the natural substrate. Expression of the anticodon nuclease core polypeptide PrrC in HeLa cells from a recombinant vaccinia virus elicited cleavage of intracellular tRNA(Lys),3. The data justify an inquiry into the possible application of anticodon nuclease as an inhibitor of tRNA(Lys),3-primed HIV replication. They also indicate that the anticodon region of tRNA(Lys) is a substrate recognition site and suggest that PrrC harbors the enzymatic activity.

POLYMORPHISM OF BACTERIAL RESTRICTION-MODIFICATION SYSTEMS: THE ADVANTAGE OF DIVERSITY.

Bacterial restriction-modification systems provide defense against foreign DNA by using a self versus nonself recognition mechanism. A great diversity of recognition motifs is maintained in natural populations. Circumstantial evidence suggests that defense against bacteriophage viruses favors this diversity. (1) Bacterial restriction enzymes can destroy invading phage DNA. (2) Phage DNA can mimic the host's self-recognition mechanism. The ability of the virus to pose as a mimic favors diversification of the host's recognition motif. Other observations suggest that restriction modification (RM) does not provide any significant defensive advantages in mature communities.

A rogue cells an indicator of cancer risk due to the action of bacterial restriction endonucleases?

Cytogenetic surveys in normal individuals have occasionally shown the occurrence of cells with multiple chromosome-type aberrations in some of the subjects. These cells, which are rare, have been termed as rogue cells. Rogue cells, which have been observed worlwide, have a mysterious nature. it has beeen suggested that they may give rise to cancer. Various mechanisms have been considered for the causation of the rouge-cell phenomenon in the past but none of them appears to be fully justified. In this paper we propose their occurrence due to the action of bacterial restriction endonucleases.

A theory that may explain the Hayflick limit — a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do — namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occured since the cells involved differentiated during development.

Success of DNA fingerprinting after failure of biotyping, antimicrobial susceptibility testing, and plasmid analysis to reveal clonality of multiple blood and urine isolates from a patient with Escherichia coli urosepsis

Multiple isolates of Escherichia coli from the blood and urine of a 60-year-old woman with acute pyelonephritis exhibited different biotypes, antimicrobial susceptibility patterns, and plasmid profiles, suggesting the presence of polymicrobial bacteriuria and leaving in question the origin of the bacteremia. Only after bacterial restriction endonuclease analysis of total bacterial DNA was it discovered that all isolates represented the same strain, with plasmid instability possibly accounting for the varied antimicrobial susceptibility patterns observed. We conclude that the biotype, antimicrobial susceptibility profile, and plasmid profile are sometimes inadequate to clarify the relationships between different clinical isolates of E. coli from a single patient and can lead to erroneous epidemiologic conclusions. DNA fingerprinting can resolve dilemmas these less precise techniques leave unresolved.

Phage introns on the hop: an alternative view

The nuclease encoded by a rare bacteriophage intron is very similar to one class of bacterial restriction enzyme, and may provide clues as to the co-evolution of host and phage.

Characterization of Chlorella virus PBCV-1 CviAII restriction and modification system.

A second DNA site-specific (restriction) endonuclease (R.CviAII) and its cognate adenine DNA methyltransferase (M.CviAII) were isolated from virus PBCV-1 infected Chlorella strain NC64A cells. R.CviAII, a heteroschizomer of the bacterial restriction endonuclease NlaIII, recognizes the sequence CATG, and does not cleave CmATG sequences. However, unlike NlaIII, which cleaves after the G and does not cleave either CmATG or mCATG sequences, CviAII cleaves between the C and A and is unaffected by mCATG methylation. The M.CviAII and R.CviAII genes were cloned and their DNA sequences were determined. These genes are tandemly arranged head-to-tail such that the TAA termination codon of the M.CviAII methyltransferase gene overlaps the ATG translational start site of R.CviAII endonuclease. R.CviAII is the first chlorella virus site-specific endonuclease gene to be cloned and sequenced.

Microflora and Bacterial DNA Restriction Enzyme Analysis in Young Adults With Periodontitis

As a result of an epidemiological survey on the periodontal status of young adults, a school was found with a relatively high number of subjects showing loss of attachment (18 out of 87). In 10 of these subjects the microflora of one pocket and of the dorsum of the tongue was investigated with special reference to black-pigmented species and Actinobacillus actinomycetemcomitans. Prevotella intermedia was the most prevalent species, being found in 9 patients. A. actinomycetemcomitans was isolated from 3 subjects and Porphyromonas gingivalis from 2 subjects. To determine whether cross-infection had occurred, the P. intermedia, P. gingivalis, and A. actinomycetemcomitans isolates were examined by restriction endonuclease analysis of the DNA after digesting with one or a combination of the endonucleases Pst I, Bam HI, and Hind III. Evaluation of the restriction endonuclease typing method showed it to be sensitive and useful for the typing of P. intermedia, P. gingivalis, and A. actinomycetemcomitans. Using this restriction endonuclease typing method, all isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia obtained from single individuals showed different digest patterns. We conclude that in the present study bacterial cross-infection did not occur among subjects in this school.

A comparative study of clinical and food isolates ofListeria monocytogenesand related species

Ninety-six isolates of presumptive or confirmed Listeria monocytogenes were obtained from local clinical (30 isolates) or food laboratories (66 isolates). Minimal biochemical analysis identified only 80% of these isolates as L. monocytogenes the remaining included L. seeligeri, 1%, or the non-haemolytic L. innocua, 19%. The 27 clinical and 50 food isolates, mainly from meat products, frozen confectionaries, and cheeses, confirmed as L. monocytogenes were compared biochemically and serologically. Twenty-one isolates, including some strains of L. innocua and L. seeligeri, were examined for pathogenicity in immunocompromized mice and 44 typed using bacterial restriction endonuclease DNA analysis (BRENDA). Only isolates of L. monocytogenes were found to be pathogenic. Biovar-typing of the isolates was unreliable and provided poor discrimination. Serogroups 1/2 and 4 predominated among clinical and food isolates and BRENDA provided better discrimination among isolates. Ten stable and reproducible restriction patterns were observed among the Listeria sp. isolates studied. Overall, a combination of techniques gave the best discrimination and indicated their potential for use as epidemiological tools.

Typing of Campylobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile

Campylobacter pylori isolates from 37 symptomatic patients and 3 asymptomatic volunteers were examined by chromosomal DNA restriction endonuclease analysis and determination of plasmid profile. Restriction digests with HindIII, HaeIII, PvuII, and BglII produced clear and reproducible results that permitted discrimination between different strains. Only 35% of C. pylori isolates were found to have plasmid DNA. Isolates from different patients, including those from two pairs of siblings, had unique restriction patterns and plasmid profiles. Consecutive isolates obtained 1 year apart from each of two asymptomatic volunteers had identical restriction patterns and plasmid profiles, suggesting persistence of the same strain. A pair of isolates obtained one year apart from the third volunteer differed in plasmid DNA content but had similar chromosomal DNA restriction patterns. Plasmid profile determination and bacterial DNA restriction endonuclease analysis provide a reliable means of discriminating between different strains of C. pylori and may be useful for typing these organisms in epidemiologic studies.

Restriction endonucleases do not induce sister-chromatid exchanges in Chinese hamster ovary cells

Bacterial restriction enzymes offer the unique opportunity to determine the biological and cytogenetic consequences of DNA double-strand breakage. To examine the role of various types of breaks in sister-chromatid exchange (SCE) formation, we used restriction enzymes with different recognition sequences and different cutting frequencies to generate DNA double-strand breaks in Chinese hamster ovary (CHO) cells. The restriction enzymes were introduced by electroporation into exponentially growing cells during the second replication cycle in bromodeoxyuridine, and SCEs were analyzed at mitosis. Contrary to results reported by others, we found no increase in SCE frequency in cells exposed to restriction enzymes despite the presence of numerous cells with chromatid aberrations. These data suggest that DNA double-strand breaks do not lead to SCE formation.

Campylobacter jejuni infection within a laboratory animal production unit

A conventional laboratory animal production unit in which rats, mice, guineapigs and rabbits were bred in one building and cats maintained in a separate, but adjacent area was examined for the presence of intestinal thermophilic Campylobacter spp. Campylobacter jejuni was recovered from 18.84% of 552 animals. The infection rate was highest amongst the cats (51.7%), with rats being the second most commonly infected (23.2%), whereas only 7.7% of guineapigs and a single rabbit (1%) were positive. Campylobacter-like organisms were cultured from 10% of the mice, but these bacteria failed to grow on subsequent subculturing. By using bacterial restriction endonuclease DNA analysis (BRENDA), a single type of C. jejuni was identified from all isolates recovered from the rats, guineapigs and a rabbit, suggesting a common source of infection. In contrast, there were 5 different BRENDA patterns derived from cat isolates. No isolates of C. jejuni were obtained from humans working within the unit or from animal bedding or the immediate environment, although it was suggested that the organism may have entered and spread within the unit from sawdust.

Seasonal prevalence of thermophilicCampylobacterinfections in dairy cattle and a study of infection of sheep

A total of 273 rectal swabs from dairy cows were cultured for Campylobacter jejuni/coli. The isolation rate was 17/72 (24%), 33/106 (31%) and 11/95 (12%) during summer, autumn and winter respectively. Approximately half of the isolates were C. jejuni and the other half C. coli. The isolates recovered from dairy cows were typed by bacterial restriction endonuclease DNA analysis (BRENDA) and compared with those of sheep. Seventeen different BRENDA patterns were produced by the isolates from dairy cows and six from 27 isolates of sheep. Of these 21 different BRENDA patterns only two were common to sheep and cattle.

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.

Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose.

Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations.

A nosocomial outbreak of Branhamella catarrhalis confirmed by restriction endonuclease analysis.

An outbreak of respiratory illness due to Branhamella catarrhalis occurred in the intermediate care unit of a Veterans Administration hospital and involved patients and staff members. Four patients had pneumonia and four had bronchitis. Infected patients were placed in a cohort separated from noninfected patients and were treated. Pharyngeal culture was used to survey prevalence in staff and all other patients on the unit; three of 18 staff members and two of 19 asymptomatic patients were positive for B. catarrhalis. A case-control study showed that respiratory therapy, steroid use, and location within the unit were significant risk factors for B. catarrhalis infection or colonization. Strains from five patients and two staff members had identical bacterial restriction endonuclease digestion patterns with three different enzymes; these patterns were distinct from those of control strains. This study is the first to document an outbreak of B. catarrhalis infection confirmed with a typing system and thus establishes B. catarrhalis as a nosocomial pathogen.