DNA Restriction Enzymes: Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1.Polymorphism, Restriction Fragment Length: Variation occurring within a species in the presence or length of DNA fragment generated by a specific endonuclease at a specific site in the genome. Such variations are generated by mutations that create or abolish recognition sites for these enzymes or change the length of the fragment.Deoxyribonucleases, Type II Site-Specific: Enzyme systems containing a single subunit and requiring only magnesium for endonucleolytic activity. The corresponding modification methylases are separate enzymes. The systems recognize specific short DNA sequences and cleave either within, or at a short specific distance from, the recognition sequence to give specific double-stranded fragments with terminal 5'-phosphates. Enzymes from different microorganisms with the same specificity are called isoschizomers. EC 3.1.21.4.Deoxyribonucleases, Type I Site-Specific: Enzyme systems containing three different subunits and requiring ATP, S-adenosylmethionine, and magnesium for endonucleolytic activity to give random double-stranded fragments with terminal 5'-phosphates. They function also as DNA-dependent ATPases and modification methylases, catalyzing the reactions of EC 2.1.1.72 and EC 2.1.1.73 with similar site-specificity. The systems recognize specific short DNA sequences and cleave at sites remote from the recognition sequence. Enzymes from different microorganisms with the same specificity are called isoschizomers. EC 3.1.21.3.DNA, Bacterial: Deoxyribonucleic acid that makes up the genetic material of bacteria.Restriction Mapping: Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.Base Sequence: The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.Deoxyribonuclease EcoRI: One of the Type II site-specific deoxyribonucleases (EC 3.1.21.4). It recognizes and cleaves the sequence G/AATTC at the slash. EcoRI is from E coliRY13. Several isoschizomers have been identified. EC 3.1.21.-.DNA: A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).Nucleic Acid Hybridization: Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503)DNA Restriction-Modification Enzymes: Systems consisting of two enzymes, a modification methylase and a restriction endonuclease. They are closely related in their specificity and protect the DNA of a given bacterial species. The methylase adds methyl groups to adenine or cytosine residues in the same target sequence that constitutes the restriction enzyme binding site. The methylation renders the target site resistant to restriction, thereby protecting DNA against cleavage.Plasmids: Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.DNA, Viral: Deoxyribonucleic acid that makes up the genetic material of viruses.Electrophoresis, Agar Gel: Electrophoresis in which agar or agarose gel is used as the diffusion medium.Deoxyribonuclease BamHI: One of the Type II site-specific deoxyribonucleases (EC 3.1.21.4). It recognizes and cleaves the sequence G/GATCC at the slash. BamHI is from Bacillus amyloliquefaciens N. Numerous isoschizomers have been identified. EC 3.1.21.-.Deoxyribonuclease HindIII: One of the Type II site-specific deoxyribonucleases (EC 3.1.21.4). It recognizes and cleaves the sequence A/AGCTT at the slash. HindIII is from Haemophilus influenzae R(d). Numerous isoschizomers have been identified. EC 3.1.21.-.Polymerase Chain Reaction: In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.DNA, Ribosomal: DNA sequences encoding RIBOSOMAL RNA and the segments of DNA separating the individual ribosomal RNA genes, referred to as RIBOSOMAL SPACER DNA.Molecular Sequence Data: Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.Caloric Restriction: Reduction in caloric intake without reduction in adequate nutrition. In experimental animals, caloric restriction has been shown to extend lifespan and enhance other physiological variables.DNA Cleavage: A reaction that severs one of the covalent sugar-phosphate linkages between NUCLEOTIDES that compose the sugar phosphate backbone of DNA. It is catalyzed enzymatically, chemically or by radiation. Cleavage may be exonucleolytic - removing the end nucleotide, or endonucleolytic - splitting the strand in two.Site-Specific DNA-Methyltransferase (Adenine-Specific): An enzyme responsible for producing a species-characteristic methylation pattern on adenine residues in a specific short base sequence in the host cell DNA. The enzyme catalyzes the methylation of DNA adenine in the presence of S-adenosyl-L-methionine to form DNA containing 6-methylaminopurine and S-adenosyl-L-homocysteine. EC 2.1.1.72.Cloning, Molecular: The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.DNA Fingerprinting: A technique for identifying individuals of a species that is based on the uniqueness of their DNA sequence. Uniqueness is determined by identifying which combination of allelic variations occur in the individual at a statistically relevant number of different loci. In forensic studies, RESTRICTION FRAGMENT LENGTH POLYMORPHISM of multiple, highly polymorphic VNTR LOCI or MICROSATELLITE REPEAT loci are analyzed. The number of loci used for the profile depends on the ALLELE FREQUENCY in the population.Genes: A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms.Blotting, Southern: A method (first developed by E.M. Southern) for detection of DNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES.Escherichia coli: A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.Bacterial Typing Techniques: Procedures for identifying types and strains of bacteria. The most frequently employed typing systems are BACTERIOPHAGE TYPING and SEROTYPING as well as bacteriocin typing and biotyping.DNA, Recombinant: Biologically active DNA which has been formed by the in vitro joining of segments of DNA from different sources. It includes the recombination joint or edge of a heteroduplex region where two recombining DNA molecules are connected.Species Specificity: The restriction of a characteristic behavior, anatomical structure or physical system, such as immune response; metabolic response, or gene or gene variant to the members of one species. It refers to that property which differentiates one species from another but it is also used for phylogenetic levels higher or lower than the species.Chromosome Mapping: Any method used for determining the location of and relative distances between genes on a chromosome.Electrophoresis, Gel, Pulsed-Field: Gel electrophoresis in which the direction of the electric field is changed periodically. This technique is similar to other electrophoretic methods normally used to separate double-stranded DNA molecules ranging in size up to tens of thousands of base-pairs. However, by alternating the electric field direction one is able to separate DNA molecules up to several million base-pairs in length.Olivomycins: A mixture of several closely related glycosidic antibiotics obtained from Actinomyces (or Streptomyces) olivoreticuli. They are used as fluorescent dyes that bind to DNA and prevent both RNA and protein synthesis and are also used as antineoplastic agents.Polymorphism, Genetic: The regular and simultaneous occurrence in a single interbreeding population of two or more discontinuous genotypes. The concept includes differences in genotypes ranging in size from a single nucleotide site (POLYMORPHISM, SINGLE NUCLEOTIDE) to large nucleotide sequences visible at a chromosomal level.RNA, Ribosomal, 16S: Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis.Deoxyribonuclease HpaII: One of the Type II site-specific deoxyribonucleases (EC 3.1.21.4). It recognizes and cleaves the sequences C/CGG and GGC/C at the slash. HpaII is from Haemophilus parainfluenzae. Several isoschizomers have been identified. EC 3.1.21.-.Sequence Analysis, DNA: A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis.Genes, Viral: The functional hereditary units of VIRUSES.Adenovirus Infections, Human: Respiratory and conjunctival infections caused by 33 identified serotypes of human adenoviruses.Genes, Bacterial: The functional hereditary units of BACTERIA.DNA, Circular: Any of the covalently closed DNA molecules found in bacteria, many viruses, mitochondria, plastids, and plasmids. Small, polydisperse circular DNA's have also been observed in a number of eukaryotic organisms and are suggested to have homology with chromosomal DNA and the capacity to be inserted into, and excised from, chromosomal DNA. It is a fragment of DNA formed by a process of looping out and deletion, containing a constant region of the mu heavy chain and the 3'-part of the mu switch region. Circular DNA is a normal product of rearrangement among gene segments encoding the variable regions of immunoglobulin light and heavy chains, as well as the T-cell receptor. (Riger et al., Glossary of Genetics, 5th ed & Segen, Dictionary of Modern Medicine, 1992)Genotype: The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS.Deoxyribonucleases, Type III Site-Specific: Enzyme systems composed of two subunits and requiring ATP and magnesium for endonucleolytic activity; they do not function as ATPases. They exist as complexes with modification methylases of similar specificity listed under EC 2.1.1.72 or EC 2.1.1.73. The systems recognize specific short DNA sequences and cleave a short distance, about 24 to 27 bases, away from the recognition sequence to give specific double-stranded fragments with terminal 5'-phosphates. Enzymes from different microorganisms with the same specificity are called isoschizomers. EC 3.1.21.5.RNA, Ribosomal: The most abundant form of RNA. Together with proteins, it forms the ribosomes, playing a structural role and also a role in ribosomal binding of mRNA and tRNAs. Individual chains are conventionally designated by their sedimentation coefficients. In eukaryotes, four large chains exist, synthesized in the nucleolus and constituting about 50% of the ribosome. (Dorland, 28th ed)Bacteriophages: Viruses whose hosts are bacterial cells.Mutation: Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.Genetic Variation: Genotypic differences observed among individuals in a population.Serotyping: Process of determining and distinguishing species of bacteria or viruses based on antigens they share.DNA Probes: Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure DNA-DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections.

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene. (1/10019)

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.  (+info)

Transposition of the autonomous Fot1 element in the filamentous fungus Fusarium oxysporum. (2/10019)

Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the so-called footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.  (+info)

Mechanisms of double-strand-break repair during gene targeting in mammalian cells. (3/10019)

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting. In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp. The hDNA was efficiently repaired prior to DNA replication. The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.  (+info)

Ataxia, ocular telangiectasia, chromosome instability, and Langerhans cell histiocytosis in a patient with an unknown breakage syndrome. (4/10019)

An 8 year old boy who had Langerhans cell histiocytosis when he was 15 months old showed psychomotor regression from the age of 2 years. Microcephaly, severe growth deficiency, and ocular telangiectasia were also evident. Magnetic nuclear resonance imaging showed cerebellar atrophy. Alphafetoprotein was increased. Chromosome instability after x irradiation and rearrangements involving chromosome 7 were found. Molecular study failed to show mutations involving the ataxia-telangiectasia gene. This patient has a clinical picture which is difficult to relate to a known breakage syndrome. Also, the relationship between the clinical phenotype and histiocytosis is unclear.  (+info)

A restriction endonuclease from Staphylococcus aureus. (5/10019)

A specific endonuclease, Sau 3AI, has been partially purified from Staphylococcus aureus strain 3A by DEAE-cellulose chromatography. The enzyme cleaves adenovirus type 5 DNA many times, SV40 DNA eight times but does not cleave double-stranded phi X174 DNA. It recognizes the sequence (see article) and cleaves as indicated by the arrows. Evidence is presented that this enzyme plays a role in the biological restriction-modification system of Staphylococcus aureus strain 3A.  (+info)

Bacillus subtilis bacteriophages SP82, SPO1, and phie: a comparison of DNAs and of peptides synthesized during infection. (6/10019)

The genomes of Bacillus subtilis phages phie, SPO1, and SP82 were compared by DNA-DNA hybridization, analysis of DNA fragments produced by digestion with restriction endonucleases, comparison of the arrays of peptides synthesized during infection, and phage neutralization. DNA-DNA hybridization experiments indicated that about 78% of the SP82 DNA was homologous with SPO1 DNA, whereas 40% of the phie DNA was homologous to either SPO1 or SP82 DNA. Agarose gel electrophoresis was used to compare the molecular weights of DNA fragments produced by cleavage of SP82, SPO1, and phie DNAs with the restriction endonucleases Hae III, Sal I, Hpa II, and Hha I. Digestion of the DNAs with Hae III and Sal I produced only a few fragments, whereas digestion with Hpa II and Hha I yielded 29 to 40 fragments, depending on the DNA and the enzyme. Comparing the Hpa II fragments, 51% of the SP82 fragments had mobilities which matched those of SPO1 fragments, 32% of the SP82 fragments matched the phie fragments, and 34% of the SPO1 fragments matched the phie fragments. Comparing the Hha I digestion products, 62% of the SP82 fragments had mobilities matching the SPO1 fragments, 24% of the SP82 fragments matched the phie fragments, and 22% of the SPO1 fragments matched the phie fragments. Analysis of peptides by electrophoresis on one-dimensional sodium dodecyl sulfate-polyacrylamide slab gels showed that approximately 70 phage-specific peptides were synthesized in the first 24 min of each infection. With mobility and the intervals of synthesis as criteria, 66% of the different SP82 peptides matched the SPO1 peptides, 34% of the SP82 peptides matched the phie peptides, and 37% of the SPO1 peptides matched the phie peptides. Phage neutralization assays using antiserum to SP82 yielded K values of 510 for SP82, 240 for SPO1, and 120 for phie.  (+info)

Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage phi105 DNA. (7/10019)

The seven previously identified EcoRI cleavage fragments of phi 105 DNA were ordered with respect to their sites of origin on the phage genome by marker rescue. One fragment, H, did not carry any determinants essential for replication. This fragment was totally missing in a deletion mutant which exhibited a lysogenization-defective phenotype. There is a nonessential region on the phi 105 genome which begins in fragment B, spans fragment H, and ends in fragment F. The size of the nonessential region, as estimated by alterations observed in the fragmentation patterns of deletion mutant DNAs, is approximately 2.7 X 10(6) daltons. Two new EcoRI cleavage fragments with molecular weights of approximately 0.2 X 10(6) were detected by autoradiography of 32P-labeled DNA. These small fragments were not located on the cleavage map.  (+info)

Restriction endonuclease mapping of bacteriophage phi105 and closely related temperate Bacillus subtilis bacteriophages rho10 and rho14. (8/10019)

Cleavage maps of the three similar Bacillus subtilis temperate bacteriophages, phi105, rho10, and rho14, were constructed by partial digestion analysis utilizing the restriction endonuclease EcoRI. Comparison of the topography of these maps indicates that all phage DNAs posses cohesive ends and a number of EcoRI restriction sites; the fragments are conserved, and the estimated base substitution/nucleotide divergence between these phages is 0.03 to 0.07 based on conserved fragments or between 0.03 and 0.11 based on conserved cleavage sites. These lines of evidence indicate that phi105, rho10, and rho14 are closely related. Double-enzyme digestion analysis reveals that rho14 DNA has unique SalGI and BglII restriction sites and phi105 DNA has a unique SalGI restriction site, making these phages possible cloning vectors for B. subtilis.  (+info)

  • Triplex stabilization by poly(L-lysine)-graft-dextran copolymer within a mammalian gene promoter inhibits the DNA binding activity of nuclear proteins from HeLa cells as well as restriction endonuclease cleavage at physiological pH and ionic conditions in vitro. (elsevier.com)
  • When the triplex-forming region (located between -165 and -146 bp) of the promoter is engineered at the Bam H1 and Pst 1 sites of a plasmid DNA, copolymer-mediated triplex stabilization also remarkably competes endonuclease activity of BamH1. (elsevier.com)
  • Our results indicate that the copolymer, regardless of the length of the target duplex, stabilizes triplexes for significant inhibition of protein - DNA interaction and endonuclease activity. (elsevier.com)
  • Ferdous, A , Akaike, T & Maruyama, A 2000, ' Inhibition of sequence-specific protein-DNA interaction and restriction endonuclease cleavage via triplex stabilization by poly(L-lysine)-graft-dextran copolymer ', Biomacromolecules , vol. 1, no. 2, pp. 186-193. (elsevier.com)
  • Electrophoretic mobility shift assays using a 30-mer hornopurine-homopyrimidine stretch (located between -170 and -141 bp) of rat α1 (I) collagen gene promoter reveal that the copolymer, at its wide range of charge ratio with DNA, stabilizes triplex DNA and enhances triplex-specific inhibition of the protein - DNA interaction. (elsevier.com)
  • Molecular cloning produces with an unlimited quantity of the DNA segments derived from a genome and these segments can be used in a range of biological sciences such as gene expression, gene therapy and so on. (lavafamilyinn.com)
  • gene (i.e.) when DNA insert is put into the lacZ? (lavafamilyinn.com)
  • galactosidase enzyme is used in genetics and molecular biology as a detection marker in gene expressions. (lavafamilyinn.com)
  • LacZ gene has a multiple cloning site within itself which can be cut by different restriction enzymes. (lavafamilyinn.com)
  • Therefore when a gene fragment is inserted into the vector, the lacZ gene is disrupted and cannot form beta-gatatosidase enzyme. (lavafamilyinn.com)
  • We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. (nih.gov)
  • Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems. (nih.gov)
  • However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures. (nih.gov)
  • Finally, some type II enzymes can generate 5 ′ overhanging DNA ends, which can only be joined to a compatible 5 ′ end. (encyclopedia.com)
  • These peptides are inactive by themselves but when both are present together they act as a functional enzyme. (lavafamilyinn.com)