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1

Monodisperse DNA restriction fragments  

Microsoft Academic Search

We present a convenient and low-cost method to prepare milligram amounts of completely monodisperse DNA restriction fragments in a physico-chemical laboratory setting to study (in part II) the effect of limited flexibility on the concentration dependent sedimentation velocity. Four fragments of 200, 400, 800, and 1600 bp were designed to span a range of 1–11 persistence lengths. The fragments were

Karel L. Planken; Gijsberta H. Koenderink; Ramon Roozendaal; Albert P. Philipse

2005-01-01

2

Transformation of Escherichia coli by a specific DNA restriction fragment  

Microsoft Academic Search

Specific transformation of a rifampicin sensitive strain of Escherichia coli to rifampicin resistance has been performed by a single, defined DNA restriction fragment carrying the genetic information for the ß subunit of E. coli RNA polymerase. In this transformation the transforming genetic character has been substituted for the corresponding recipient gene locus by recombination. The value of the described transformation

Silvia M. Schweitzer; Hans Matzura

1977-01-01

3

Discriminatory 32P 3'-end labeling of restriction endonuclease co-digested DNA fragments.  

PubMed

We present an improved method for selectively labeling specific DNA fragments in a mixture of restriction fragments. lambda DNA, used to develop the procedure, was digested with a combination of restriction enzymes and treated, at various incubation temperatures, with reverse transcriptase and one or more [alpha-32P]dNTP molecules. The labeled fragments were subjected to agarose gel electrophoresis and detected by autoradiography. We were able to direct the 3'-end labeling to precise subpopulations of DNA fragments, generated by multiple restriction enzyme digestions. We also show that labeling selectivity of DNA restriction fragments, by reverse transcriptase, is affected by the incubation temperature used during the labeling reaction. This paper describes an approach to 3'-end label select DNA fragments with reverse transcriptase and [alpha-32P]dNTPs. The procedure permits the bypassing of time-consuming isolation and purification steps required, by conventional radiolabeling techniques, to radiolabel specific DNA fragments. PMID:2543232

LeBlond, G F; Ts'o, P O

1989-03-01

4

The Restriction Fragment Map of Rat-Liver Mitochondrial DNA: A Reconsideration  

Microsoft Academic Search

1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Bam HI and 11 for the restriction endonuclease Hap II.\\u000a2. The physical map of the restriction fragments of Eco RI, Hind III, Bam HI and Hap II is constructed on

A. M. Kroon; G. Pepe; H. Bakker; M. Holtrop; J. E. Bollen; E. F. J. van Bruggen; P. Cantatore; P. Terpstra; C. Saccone

1977-01-01

5

Hypervariable Bkm DNA Loci in a Moth, Ephestia kuehniella: Does Transposition Cause Restriction Fragment Length Polymorphism?  

Microsoft Academic Search

Bkm sequences, originally isolated from snake satellite DNA, are a component of eukaryote genomes with a preferential location on sex chromosomes. In the Ephestia genome, owing to the presence of only a few Bkm-positive BamHI restriction fragments and to extensive restriction fragment length polymorphisms between and within inbred strains, a genetic crossbreeding analysis was feasible. No sex linkage of Bkm

W. Traut; Accepted December

1987-01-01

6

Cytoplasmic DNAs and nuclear rDNA restriction fragment length polymorphisms in commercial witloof chicories  

Microsoft Academic Search

Restriction fragment length polymorphisms of cytoplasmic DNAs and nuclear rDNA were analyzed in several Cichorium intybus genotypes, comprising four white inbred lines, eight red witloof experimental lines, and a number of F1 hybrids derived from two white parents. Chloroplast and mitochondrial restriction patterns led to the distinction between two different cytoplasms, called I and II. Southern hybridization using a nuclear

A. Bellamy; C. Mathieu; F. Vedel; H. Bannerot

1995-01-01

7

DNA restriction fragment length polymorphisms and heterozygosity in the human genome  

Microsoft Academic Search

A list is presented of published reports of DNA polymorphisms found in the human genome by restriction enzyme analysis. While the list indicates the large number of restriction fragment length polymorphisms (RFLPs) detected to date, the information collated is insufficient to permit an estimate of heterozygosity for the genome as a whole. Data from our laboratory are therefore also presented

David N. Cooper; Jörg Schmidtke

1984-01-01

8

DNA restriction fragment analysis of the proopiomelanocortin gene in schizophrenia and bipolar disorders.  

PubMed Central

The method of DNA restriction fragment analysis using gene probes for the proopiomelanocortin (POMC) gene was employed to detect possible molecular variation in the POMC gene in schizophrenia and bipolar illness. No gross structural abnormalities in restriction fragments were observed with the set of restriction enzymes used. Two allelic restriction sites were observed giving rise to fragment length polymorphisms. One of these is a new polymorphism, not previously reported, which will be of value as a linkage marker. The associations between the two DNA polymorphisms that are closely linked to the POMC gene and both schizophrenia and bipolar disorder were investigated. No association was found, thus adding weight to the evidence that there are no alterations in the POMC gene in schizophrenia and bipolar illness. Images Fig. 1

Feder, J; Gurling, H M; Darby, J; Cavalli-Sforza, L L

1985-01-01

9

DNA restriction-fragment variation in the gene family encoding high molecular weight (HMW) glutenin subunits of wheat  

Microsoft Academic Search

Restriction enzyme digests of DNA from nullisomic-tetrasomic and intervarietal chromosome substitution lines of wheat were probed with a high molecular weight (HMW) glutenin cDNA. Three restriction endonucleases were used to investigate restriction-fragment differences among five wheat varieties. The results suggest that the hybridizing fragments contain single gene copies and permit the identification of the subunit encoded by each gene. Restriction-fragment

N. P. Harberd; D. Bartels; R. D. Thompson

1986-01-01

10

Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting  

Microsoft Academic Search

BACKGROUND: Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance), that calculates the relative abundance of genes in cDNA libraries. RESULTS: DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of

Peter Hof; Claudia Ortmeier; Kirstin Pape; Birgit Reitmaier; Johannes Regenbogen; Andreas Goppelt; Joern-Peter Halle

2002-01-01

11

High interindividual restriction fragment length and copy number of polymorphism of a TVRI family in moderate human DNA repeats  

SciTech Connect

The authors describe the selection of cloned human DNA sequences, with a copy number not exceeding 1000 copies per diploid genome, and their testing for interindividual restriction fragment lengths and copy number of polymorphism (RFLCP). As a result of the investigation a DNA clone was found (TVRI-6), about 2.8 kilobase-pairs in size, for which an unusually high level of interindividual RFLCP was discovered. The TVRI-6 sequence was obtained from a bank of Pst I restriction fragments of human placental nuclear DNA cloned in pBR 322. The bank was analyzed by hybridization of colonies with phosphorus 32-labelled human nuclear DNA.

Rogaev, E.I.; Shapiro, Yu.A.

1987-06-01

12

Capillary electrophoretic separation of DNA restriction fragments using dilute polymer solutions  

SciTech Connect

Because the mechanism of DNA separation in capillary electrophoresis is not well understood, selection of polymers is a {open_quotes}trial-and-error{close_quotes} procedure. We investigated dilute-solution DNA separations by capillary electrophoresis using solutions of four polymers that differ in size, shape and stiffness. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp - 23130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp and star-(polyethylene oxide), like linear poly (ethylene oxide), provides separation of fragments up to 1353 bp.

Braun, B.; Blanch, W.; Prausnitz, J.M.

1997-02-01

13

Mitochondrial dna restriction fragment length polymorphisms in fusarium oxysporum f. sp. niveum  

Microsoft Academic Search

Mitochondrial DNA (mtDNA) extracts from 13 isolatesof Fusarium oxysporum f. sp.niveum, including 12 from widely separated geographic regions within the United States and representing the three races, and one\\u000a race 2 isolate from Israel, were examined for the presence of plasmid DNA and were also subjected to restriction endonucleases\\u000a analysis. None of the mtDNA from any isolate had a copurifying

D. H. kim; R. D. martyn; C. W. magill

1991-01-01

14

Single-Molecule Analysis of Restriction DNA Fragments Using Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

The cleavage of fluorescence-labeled M13DNA (7250 bp) usingHaeIII,HgaI,BsmAI, andBspMI was analyzed by fluorescence correlation spectroscopy (FCS) in a small volume (1.5 × 10?15liters). The digestion process can be monitored by the decrease in amplitude of the fluorescence correlation function while the original DNA molecule is divided into several fragments by the enzymes. To analyze this reaction by FCS, we derived

Masataka Kinjo; Goro Nishimura; Tomiyasu Koyama; Ülo Mets; Rudolf Rigler

1998-01-01

15

Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens).  

PubMed

A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment-length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist. PMID:2895887

Quinn, T W; White, B N

1987-03-01

16

Characterization of Escherichia hermannii by ribosomal DNA restriction fragment length polymorphism.  

PubMed

Ribosomal DNA polymorphism was used to characterize strains of Escherichia hermannii and to differentiate them from E. coli. DNA from 11 E. hermannii strains previously separated into three zymotypes by enzyme electrophoretic polymorphism was digested with HindIII and EcoRI restriction enzymes and analyzed by Southern blotting. The 10 ribotypes obtained with EcoRI fell into 3 groups which correlated with the corresponding zymotypes, and the 5 ribotypes obtained with HindIII were clearly distinct from those of E. coli strains. PMID:7910695

Picard-Pasquier, N; Picard, B; Krishnamoorthy, R; Goullet, P

17

Investigation of hospital-acquired infections due to Alcaligenes denitrificans subsp. xylosoxydans by DNA restriction fragment length polymorphism.  

PubMed Central

We demonstrate that DNA restriction fragment length polymorphism determined by pulsed-field gel electrophoresis is very useful in the investigation of the epidemiology of hospital-acquired infections caused by Alcaligenes denitrificans subsp. xylosoxydans. This approach showed that hospital-acquired infections caused by this opportunistic pathogen over a 6-month period in 10 patients hospitalized in an intensive care unit and a surgical unit were not a true outbreak. In addition, this molecular typing method established that the respiratory therapy equipment was the source of the contamination of two patients. Images

Cheron, M; Abachin, E; Guerot, E; el-Bez, M; Simonet, M

1994-01-01

18

Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments.  

PubMed Central

Gapped duplex DNA molecules of recombinant genomes of filamentous phage are constructed in vitro. Denatured restriction fragments covering (part of) the precisely constructed gap are hybridized to the gapped duplex DNA molecules to form ternary duplices. The two strands of the ternary duplex molecules carry different genetic markers within the region spanned by the restriction fragment leading to a one base pair mismatch or to an insertion loop of 93 nucleotides, respectively. The two strands also vary with respect to A-methylation in GATC sites. In cases of asymmetrical methylation, transfection of E. coli with these heteroduplex molecules leads to marker recoveries with a pronounced bias in favour of the marker encoded by the methylated strand. This effect at least partly explains the comparably low marker yields achieved in previous directed mutagenesis experiments using filamentous phage as the vector. The results suggest how these procedures can be optimized. Precise construction of a 93 bp insertion of 9.5% marker yield is described.

Kramer, W; Schughart, K; Fritz, H J

1982-01-01

19

Novel Approach to the Ligation of Single-Stranded DNA Fragments by T4 DNA Ligase—DNA Mobile Multiple-Restriction Fragments: “UNI-LINKERS” for Cloning of Genes  

Microsoft Academic Search

A group of uniquely designed single-stranded oligodeoxyribo-nucleotides that form hairpin loops were synthesized. These oligonucleotides can be ligated to other synthetic single-stranded fragents differing in length and design without the need for external annealing templates. A novel approach to building a limitless variety of mobile multiple-restriction DNA fragments termed “uni-linkers” and which are open only at one end, is described.

Fawzy Georges; Ravindra N. Chibbar; W. Jay Newsted; Friedrich Constabel

1989-01-01

20

Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism Analysis of dnaJ Gene?  

PubMed Central

A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.

Hauschild, Tomasz; Stepanovic, Srdjan

2008-01-01

21

Evaluation of restriction fragment length polymorphism analysis of 16S rDNA as a tool for genomovar characterisation within the Burkholderia cepacia complex  

Microsoft Academic Search

A total of 154 Burkholderia cepacia complex strains, isolated from cystic fibrosis and non-cystic fibrosis patients and the environment, representing all nine genomovars and a putative tenth, were analysed by 16S rDNA-restriction fragment length polymorphism using the restriction enzymes AluI, CfoI and DdeI. Examining this diverse strain collection resulted in very diverse restriction patterns. Only B. cepacia genomovar VI could

Karen Vermis; Christoph Vandekerckhove; Hans J Nelis; Peter A. R Vandamme

2002-01-01

22

Kinetics of circular DNA molecule digestion by restriction endonuclease Computation of kinetic constants from time dependence of fragment concentrations  

Microsoft Academic Search

A model for kinetics of circular substrate cleavage by restriction endonuclease was formulated. The aim of the analysis of the model was to extract kinetic constants for all target sites from time-dependence of fragment concentration in reaction products. That was proved to be possible for molecules with an odd number of fragments only. A symmetry of the molecules with an

Petr Karlovský

1986-01-01

23

Restriction Fragment Length Polymorphism Analysis of Ribosomal DNA Intergenic Regions Is Useful for Differentiating Strains of Trichophyton mentagrophytes  

PubMed Central

Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates.

Mochizuki, Takashi; Ishizaki, Hiroshi; Barton, Richard C.; Moore, Mary K.; Jackson, Colin J.; Kelly, Steven L.; Evans, E. Glyn V.

2003-01-01

24

Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L  

Microsoft Academic Search

CpDNA variation among 52 tree samples belonging to 25 different taxa of Coffea and two species of Psilanthus was assessed by RFLP analysis on both the total chloroplast genome and the atpB-rbcL intergenic region. Twelve variable characters were distinguished allowing the identification of 12 different plastomes. The low sequence divergence observed might suggest that Coffea is a young genus. The

P. Lashermes; J. Cros; M. C. Combes; P. Trouslot; F. Anthony; S. Hamon; A. Charrier

1996-01-01

25

Evaluation of restriction fragment length polymorphism analysis of 16S rDNA as a tool for genomovar characterisation within the Burkholderia cepacia complex.  

PubMed

A total of 154 Burkholderia cepacia complex strains, isolated from cystic fibrosis and non-cystic fibrosis patients and the environment, representing all nine genomovars and a putative tenth, were analysed by 16S rDNA-restriction fragment length polymorphism using the restriction enzymes AluI, CfoI and DdeI. Examining this diverse strain collection resulted in very diverse restriction patterns. Only B. cepacia genomovar VI could be identified unambiguously. The same restriction patterns were observed for B. cepacia genomovars I and III and approximately half of the Burkholderia ambifaria, B. anthina and B. pyrrocinia strains. Burkholderia vietnamiensis and B. ubonensis, a putative tenth B. cepacia complex genomovar, shared identical restriction profiles. The majority of Burkholderia multivorans and B. stabilis isolates generated a unique restriction pattern, but two strains of each showed divergent restriction profiles which were also observed in other genomovars. PMID:12204364

Vermis, Karen; Vandekerckhove, Christoph; Nelis, Hans J; Vandamme, Peter A R

2002-08-27

26

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization.  

PubMed

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities. PMID:21824243

Wang, Shanquan; He, Jianzhong

2011-08-08

27

New strain typing method with Sporothrix schenckii using mitochondrial DNA and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique.  

PubMed

The complete sequences of mitochondrial DNA (mtDNA) from two strains of different genotypes, American Type Culture Collection 10268 of mtDNA type 1 and KMU2025 of mtDNA type 4, were determined. These are circular molecules, 27 125 and 26 095 bp in length, respectively. The greatest difference between the two strains was found in the region encompassed by atp9 and cox2 genes, which was amplified with polymerase chain reaction (PCR) and used for preliminary restriction fragment length polymorphism (RFLP) analysis. Eight isolates of five mtDNA types were used and RFLP patterns obtained with the restriction enzyme AseI showed that this method seems to have greater discrimination power than the other PCR-RFLP typing method using internal transcribed spacer regions of nuclear DNA. PMID:21955258

Kawasaki, Masako; Anzawa, Kazushi; Mochizuki, Takashi; Ishizaki, Hiroshi

2011-09-28

28

Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA.  

PubMed Central

The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.

Williams, D W; Wilson, M J; Lewis, M A; Potts, A J

1995-01-01

29

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages.  

PubMed Central

Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate. Images

Yolov, A A; Gromova, E S; Kubareva, E A; Potapov, V K; Shabarova, Z A

1985-01-01

30

Restriction Fragment Length Polymorphism Linkage Map for Arabidopsis thaliana  

Microsoft Academic Search

We have constructed a restriction fragment length polymorphism linkage map for the nuclear genome of the flowering plant Arabidopsis thaliana. The map, containing 90 randomly distributed molecular markers, is physically very dense; >50% of the genome is within 1.9 centimorgans, or ≈ 270 kilobase pairs, of the mapped DNA fragments. The map was based on the meiotic segregation of markers

Caren Chang; John L. Bowman; Arthur W. Dejohn; Eric S. Lander; Elliot M. Meyerowitz

1988-01-01

31

Stock Structure and Homing Fidelity in Gulf of Mexico Sturgeon (Acipenser Oxyrinchus Desotoi) Based on Restriction Fragment Length Polymorphism and Sequence Analyses of Mitochondrial DNA  

PubMed Central

Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FL, (3) Choctawhatchee River, FL, and (4) Apalachicola, Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids.

Stabile, J.; Waldman, J. R.; Parauka, F.; Wirgin, I.

1996-01-01

32

Stock structure and homing fidelity in Gulf of Mexico sturgeon (Acipenser oxyrinchus desotoi) based on restriction fragment length polymorphism and sequence analyses of mitochondrial DNA.  

PubMed

Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FI, (3) Choctawbatchee River, FL and (4) Apalachicola Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids. PMID:8889537

Stabile, J; Waldman, J R; Parauka, F; Wirgin, I

1996-10-01

33

Restriction fragment length polymorphism of 195 bp repeated satellite dna of Trypanosoma cruzi supports the existence of two phylogenetic groups  

Microsoft Academic Search

Hinf I) and high molecular weight DNA (Hae III), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by isoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic

Brigitte Bastrenta; Marie France Bosseno; Christian Barnabé; Michel Tibayrenc; Simone Frédérique Breničre

1999-01-01

34

Different restriction fragment pattern of mtDNA indicative of generalized 8993 point mutations in a boy with lactic acidosis  

Microsoft Academic Search

A mitochondrial DNA (mtDNA) T-G transversion at 8993 associated with NARP syndrome (neurogenic muscle weakness, ataxia, retinitis pigmentosa) was identified by Holt et al (1990). The same mutation was detected in two families with maternally inherited Leigh syndrome by Tatuch et al (1992) and Ciafaloni et al (1993). Heteroplasmy was shown to play an important role in the clinical phenotype,

P. Klement; J. Zeman; H. Hansikova; H. Houstkova; M. Baudysova; J. Houstek

1994-01-01

35

Mapping Flagellar Genes in Chlamydomonas Using Restriction Fragment Length Polymorphisms  

Microsoft Academic Search

To correlate cloned nuclear DNA sequences with previously characterized mutations in Chlamy- domonas and, to gain insight into the organization of its nuclear genome, we have begun to map molecular markers using restriction fragment length polymorphisms (RFLPs). A Chlamydomonas reinhardtii strain (CC-29) containing phenotypic markers on nine of the 19 linkage groups was crossed to the interfertile species Chlamydomonas smithii.

Laura P. W. Ranum; Michael D. Thompson; Jeffery A. Schloss; Paul A. Lefebvre; Carolyn D. Silflow

1988-01-01

36

Restriction fragment polymorphism in the sex-determining region of the Y chromosomal DNA of European wild mice  

Microsoft Academic Search

Using 32P-labeled probe consisting mainly of (GATA)n we have shown that a male specific Alu1 DNA blot pattern which defines the Y chromosome sex-determining locus in inbred mice is highly polymorphic in wild mice, indicating substantial sequence evolution in this region under field conditions. In all cases examined by in situ hybridization, the region concerned is paracentromeric. In contrast, the

L. Singh; H. Winking; K. W. Jones; A. Gropp

1988-01-01

37

Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes  

NASA Astrophysics Data System (ADS)

This study explores the applicability of DNA analyses for the characterization of primary biogenic aerosol (PBA) particles in the atmosphere. Samples of fine particulate matter (PM2.5) and total suspended particulates (TSP) have been collected on different types of filter materials at urban, rural, and high-alpine locations along an altitude transect in the south of Germany (Munich, Hohenpeissenberg, Mt. Zugspitze). From filter segments loaded with about one milligram of air particulate matter, DNA could be extracted and DNA sequences could be determined for bacteria, fungi, plants and animals. Sequence analyses were used to determine the identity of biological organisms, and terminal restriction fragment length polymorphism analyses (T-RFLP) were applied to estimate diversities and relative abundances of bacteria. Investigations of blank and background samples showed that filter materials have to be decontaminated prior to use, and that the sampling and handling procedures have to be carefully controlled to avoid artifacts in the analyses. Mass fractions of DNA in PM2.5 were found to be around 0.05% in urban, rural, and high-alpine aerosols. The average concentration of DNA determined for urban air was on the order of ~7 ng m-3, indicating that human adults may inhale about one microgram of DNA per day (corresponding to ~108 haploid bacterial genomes or ~105 haploid human genomes, respectively). Most of the bacterial sequences found in PM2.5 were from Proteobacteria (42) and some from Actinobacteria (10) and Firmicutes (1). The fungal sequences were characteristic for Ascomycota (3) and Basidiomycota (1), which are known to actively discharge spores into the atmosphere. The plant sequences could be attributed to green plants (2) and moss spores (2), while animal DNA was found only for one unicellular eukaryote (protist). Over 80% of the 53 bacterial sequences could be matched to one of the 19 T-RF peaks found in the PM2.5 samples, but only 40% of the T-RF peaks did correspond to one of the detected bacterial sequences. The results demonstrate that the T-RFLP analysis covered more of the bacterial diversity than the sequence analysis. Shannon-Weaver indices calculated from both sequence and T-RFLP data indicate that the bacterial diversity in the rural samples was higher than in the urban and alpine samples. Two of the bacterial sequences (Gammaproteobacteria) and five of the T-RF peaks were found at all sampling locations.

Després, V. R.; Nowoisky, J. F.; Klose, M.; Conrad, R.; Andreae, M. O.; Pöschl, U.

2007-12-01

38

Genetic Diversity among Strains of Moraxella catarrhalis: Analysis Using Multiple DNA Probes and a Single-Locus PCR-Restriction Fragment Length Polymorphism Method  

PubMed Central

Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce ?-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.

Walker, Elaine S.; Preston, Robert A.; Post, J. Christopher; Ehrlich, Garth D.; Kalbfleisch, John H.; Klingman, Karin L.

1998-01-01

39

Analysis of mitochondrial DNA for authentication of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) by polymerase chain reaction-restriction fragment length polymorphism.  

PubMed

The prevention of fraudulent labeling of game meat constitutes an important part of food regulatory control and quality assurance systems. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis based on mitochondrial deoxyribonucleic acid (DNA) was developed for authentication of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon). Amplification and restriction site analysis of a DNA fragment about 720 base pairs (bp) from the mitochondrial 12S rRNA gene of all analyzed species permitted the selection of Msel and Apol endonucleases for meat speciation. The 12S rRNA restriction profiles obtained allowed the unequivocal identification of chamois, pyrenean ibex, and mouflon/sheep and their differentiation from meats of domestic species such as cattle, goat, and swine. The highly variable mitochondrial D-loop gene was also targeted to attempt discrimination between mouflon and sheep meats. A D-loop region (700-1000 bp) was amplified and sequenced in all game and domestic species analyzed, and a primer set was designed for the selective amplification of a 370 bp DNA fragment from mouflon and sheep. PCR-RFLP analysis with the selected Maell enzyme generated a single electrophoretic profile characteristic for sheep, whereas 3 different fragment patterns were obtained for mouflon meats. Consequently, the PCR-RFLP technique developed can be routinely applied in inspection programs in order to verify the correct labeling of game species. PMID:17373450

Fajardo, Violeta; González, Isabel; López-Calleja, Inés; Martin, Irene; Rojas, Maria; Pavón, Miguel Angel; Hernández, Pablo E; García, Teresa; Martín, Rosario

40

Restriction fragment length polymorphisms of 16S rDNA and of whole rRNA genes (ribotyping) of Streptococcus iniae strains from the United States and Israel  

Microsoft Academic Search

Streptococcus iniae (junior synonym S. shiloi) isolated from tilapia and trout in Israel and in the United States were subtyped by restriction length polymorphism (RFLP) based on PCR amplified 16S rDNA and by ribotyping. 16S rDNA RFLP discriminated between S. iniae and other fish pathogens but not between S. iniae strains. HindIII and EcoRI ribotypes of S. iniae discriminated American

Avi Eldar; Sara Lawhon; Paul F. Frelier; Liliana Assenta; Bruce R Simpson; Patricia W Varner; Herve Bercovier

1997-01-01

41

Genetic Relationships among Populations of the Senegalese Sole Solea senegalensis in the Southwestern Iberian Peninsula Detected by Mitochondrial DNA–Restriction Fragment Length Polymorphisms  

Microsoft Academic Search

We assessed the population structure of the Senegalese sole Solea senegalensis in the southern Atlantic off the coast of the Iberian peninsula using restriction fragment length polymorphism analysis of three mitochondrial gene regions: 16S rRNA (620 base pairs [bp]), 12S rRNA (440 bp), and cytochrome b (380 bp). Fourteen composite haplotypes were detected among 109 adult fish from five natural

Edgardo Díaz-Ferguson; Ismael Cross; Maria Mar Del Barrios; Laureana Rebordinos

2007-01-01

42

Taxonomy and phylogeny of some Eimeria (Apicomplexa: Eimeriidae) species of rodents as determined by polymerase chain reaction\\/restriction-fragment-length polymorphism analysis of 18S rDNA  

Microsoft Academic Search

The 18S rDNA genes of 10 Eimeria species from rodents (E. albigulae, E. arizonensis, E. falciformis, E. langebarteli, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis) were polymerase-chain-reaction (PCR)-amplified, digested with 12 restriction endonucleases, and electophoresed in agarose\\u000a gels. The resulting fragment patterns (riboprints) distinguished all species except E. sevilletensis from E.?falciformis, and E. arizonensis from

J. A. Hnida; D. W. Duszynski

1999-01-01

43

Capillary electropherograms for restriction fragment length polymorphism of Helicobacter pylori.  

PubMed

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains. PMID:18958869

Bair, Ming-Jong; Chen, Chiu-Lin; Chiang, Cheng-Kang; Huang, Ming-Feng; Hu, Cho-Chun; Chang, Huan-Tsung

2008-10-01

44

Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms  

Microsoft Academic Search

Genetic linkage maps were constructed for both maize and tomato, utilizing restriction fragment length polymorphisms (RFLPs) as the source of genetic markers. In order to detect these RFLPs, unique DNA sequence clones were prepared from either maize or tomato tissue and hybridized to Southern blots containing restriction enzyme-digested genomic DNA from different homozygous lines. A subsequent comparison of the RFLP

T. Helentjaris; M. Slocum; S. Wright; A. Schaefer; J. Nienhuis

1986-01-01

45

Genetic evidence for the existence of cryptic species in the Anopheles albitarsis complex in Brazil: Allozymes and mitochondrial DNA restriction fragment length polymorphisms  

Microsoft Academic Search

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among\\u000a field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types\\u000a of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2)

S. K. Narang; T. A. Klein; O. P. Perera; José Bento Lima; Amazonia Toda Tang

1993-01-01

46

Physical mapping of Bgl II, Bam HI, Eco RI, Hin dIII and Pst I Restriction fragments of bacteriophage P1 DNA  

Microsoft Academic Search

A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII (3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order

Brigitte Bächi; Werner Arber

1977-01-01

47

Sizing of single fluorescently stained DNA fragments by scanning microscopy  

PubMed Central

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-l-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7–14%. The proposed method is straightforward and can be applied to standardized microtiter plates.

Laib, Stephan; Rankl, Michael; Ruckstuhl, Thomas; Seeger, Stefan

2003-01-01

48

MspI restriction fragment length polymorphism at the glycoprotein hormone ?-subunit locus  

Microsoft Academic Search

A restriction fragment length polymorphism (RFLP) in the human glycoprotein hormone common ?-subunit gene has been identified and partially characterized in normal lymphocytes and placentae, established tumor cell lines, and tumor biopsy samples. High molecular weight DNA was digested with the restriction endonuclease MspI, separated by electrophoresis in agarose gels, transferred to nylon membranes by the method of Southern, and

G. Stanley Cox; Dominic E. Cosgrove; Michael J. Haas; Warren Stiles; David G. McIntosh

1997-01-01

49

The linkage mapping of cloned restriction fragment length differences in Caenorabditis elegans  

Microsoft Academic Search

The genomic DNA of two closely related strains of the nematode, Caenorhabditis elegans, Bristol (N2), and Bergerac (Bo), has different restriction endonuclease sites (Emmons et al. 1979). Since these two strains interbreed, it is possible to regard the restriction fragment length differences (RFLDs) as mutant variants. The N2 and Bo pattern can be segregated and mapped using clasical genetic techniques.

A. M. Rose; D. L. Baillie; E. P. M. Candido; K. A. Beckenbach; D. Nelson

1982-01-01

50

Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes.  

PubMed

Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13 x 10(8) cfu g(-1) feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO(2) and NH(3) evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts. PMID:15614566

Tiquia, S M; Ichida, J M; Keener, H M; Elwell, D L; Burtt, E H; Michel, F C

2004-12-22

51

DNA Fragmentation in Microorganisms Assessed In Situ?  

PubMed Central

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions.

Fernandez, Jose Luis; Cartelle, Monica; Muriel, Lourdes; Santiso, Rebeca; Tamayo, Maria; Goyanes, Vicente; Gosalvez, Jaime; Bou, German

2008-01-01

52

Characterization of Bacterial Community Diversity in Cystic Fibrosis Lung Infections by Use of 16S Ribosomal DNA Terminal Restriction Fragment Length Polymorphism Profiling  

PubMed Central

Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial community in 71 sputa from 34 adult CF patients. Nineteen species from 15 genera were identified in 53 16S rRNA clones from three patients. Of these, 15 species have not previously been reported in CF lung infections and many were species requiring strict anaerobic conditions for growth. The species richness and evenness were determined from the T-RF length and volume for the 71 profiles. Species richness was on average 13.3 ± 7.9 per sample and 13.4 ± 6.7 per patient. On average, the T-RF bands of the lowest and highest volumes represented 0.6 and 59.2% of the total volume in each profile, respectively. The second through fifth most dominant T-RF bands represented 15.3, 7.5, 4.7, and 2.8% of the total profile volume, respectively. On average, the remaining T-RF bands represented 10.2% of the total profile volume. The T-RF band corresponding to Pseudomonas aeruginosa had the highest volume in 61.1% of the samples. However, 18 other T-RF band lengths were dominant in at least one sample. In conclusion, this reveals the enormous complexity of bacteria within the CF lung. Although their significance is yet to be determined, these findings alter our perception of CF lung infections.

Rogers, G. B.; Carroll, M. P.; Serisier, D. J.; Hockey, P. M.; Jones, G.; Bruce, K. D.

2004-01-01

53

Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis  

Microsoft Academic Search

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which

Ivo Pavlik; Alica Horvathova; Lenka Dvorska; Jiri Bartl; Petra Svastova; Robin du Maine; Ivan Rychlik

1999-01-01

54

Restriction fragment analysis of bacteriophage SPP1 in vitro transcription by host RNA polymerase.  

PubMed Central

In vitro transcription of SPP1 DNA occurred on only one of the two strands, the same which is predominantly transcribed in SPP1-infected cells. Transcripts were distributed in several size classes. Analysis of elongation kinetics and of size distribution, coupled with hybridization to DNA restriction fragments, showed that some regions of the template have more initiation sites than others; some have none. Some regions were transcribed directly, some were transcribed from initiation sites located in other regions, and one was never transcribed. Several transcription initiation sites on SPP1 DNA are located on EcoRI fragment 1; four to five others are distributed among other fragments. Cutting the DNA with EcoRI did not introduce artifactual initiation sites. In vitro transcription units can be localized and oriented with respect to the EcoRI restriction map of SPP1 DNA. Images

Chenciner, N; Milanesi, G

1978-01-01

55

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers  

Microsoft Academic Search

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a poly- merase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in

G. Yan; J. Romero-Severson; M. Walton; D. D. CHADEEand; D. W. Severson

1999-01-01

56

Specific Cleavage of Simian Virus 40 DNA by Restriction Endonuclease of Hemophilus Influenzae  

Microsoft Academic Search

A bacterial restriction endonuclease has been used to produce specific fragments of SV40 DNA. Digestion of DNA from plaque-purified stocks of SV40 with the restriction endonuclease from Hemophilus influenzae gave 11 fragments resolvable by polyacrylamide gel electrophoresis, eight of which were equimolar with the original DNA. The fragments ranged from about 6.5 × 105 to 7.4 × 104 daltons, as

Kathleen Danna; Daniel Nathans

1971-01-01

57

Direct endonuclease digestion and multi-analysis of restriction fragment length polymorphisms by microchip electrophoresis  

Microsoft Academic Search

A high-performance multi-analysis system for genotypic mutation by means of restriction fragment length polymorphisms (RFLP) involving endonuclease treatment of PCR-amplified DNA on a microchip and subsequent analysis by microchip electrophoresis for DNA sizing was developed. A Hitachi SV1210 system, with which 12 samples can be analyzed on a plastic chip with good accuracy as to DNA sizing between 25 and

Rie Akamine; Shouki Yatsushiro; Shouhei Yamamura; Jun-ichi Kido; Yasuo Shinohara; Yoshinobu Baba; Masatoshi Kataoka

2009-01-01

58

A rapid and efficient method for the isolation of mitochondrial DNA from angiosperm tree species. Application to the restriction fragment length polymorphism distinction between European and American ashes  

Microsoft Academic Search

A simplified method for the isolation of mitochondrial DNA (mtDNA) from several angiosperm tree species is described. The procedure does not require gradient ultracen- trifugation or organic solvent extractions and can be performed in a single day. mtDNA was isolated from flower buds collected from adult trees just after the first pollen mitosis and purified by gravity flow through disposable

M. E. Morand; F. Vedel; C. Raquin; N. Frascaria-Lacoste

2001-01-01

59

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level  

Microsoft Academic Search

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA

Yuichi Michikawa; Keisuke Sugahara; Tomo Suga; Yoshimi Ohtsuka; Kenichi Ishikawa; Atsuko Ishikawa; Naoko Shiomi; Tadahiro Shiomi; Mayumi Iwakawa; Takashi Imai

2008-01-01

60

Clusters of HLA class II beta restriction fragments describe allelic series.  

PubMed Central

Eighty-eight HLA haplotypes have been investigated for the presence or absence of 52 restriction fragments generated by four restriction enzymes (EcoRI, EcoRV, HindIII, BamHI), and detected by a DQ beta cDNA probe. Correlation analysis showed several sets of positively associated fragments forming 11 clusters. They constitute three different allelic series. The first coincides with DR alleles, the second with DQ alleles, and within the third, one cluster coincides with DRw53 (MT). As shown by comparative hybridization, most fragments belonging to the DR- as well as the DQ-related series correspond to DQ beta genes. In contrast, the MT-related series corresponds to DR beta genes. The evolutionary significance of these restriction fragment clusters is discussed. Images

Cohen, D; Le Gall, I; Marcadet, A; Font, M P; Lalouel, J M; Dausset, J

1984-01-01

61

Molecular identification of fecal pollution sources in water supplies by host-specific fecal DNA markers and Terminal Restriction Fragment Length Polymorphism profiles of 16S rRNA gene.  

PubMed

Specific fecal DNA markers were investigated for major pollution sources, cow, human, and pig, and occurrence of the identified markers was analyzed in river waters using Terminal Restriction Fragment Length Polymorphism (T-RFLP) techniques and sequencing of 16S rDNA of Bacteroides-Prevotella. The unique and specific DNA markers for cow and human were identified as a 222 bp and 60 bp peak in HaeIII T-RFLP profiles, respectively, and the pig-specific marker was not identified but the unique T-RFLP profile of pig could be used as a substitution. Human-specific marker was detected in most of the river waters tested (92.1%) and T-RFLP profiles of river waters were shown to be similar to those of human feces. Cluster analysis of T-RFLP data showed that the fecal sources were multiple (human plus cow and human plus dairy cow) in most of the river waters. The phylogenetic analysis for the clones recovered from the fecal and water samples showed that the clones from cow formed a discreet cluster from those of other sources. The other clones from human, pig, and river water formed two groups all together. The results of this study could be used to identify and control the fecal pollution source in the bodies of water in Korea. PMID:19107387

Jeong, Ju-Yong; Gil, Kyung-Ik; Lee, Kyong-Hee; Ka, Jong-Ok

2008-12-24

62

Restriction fragments homologous to mitochondrial plasmid-like DNAs are located within limited chromosomal regions on the rice nuclear genome  

Microsoft Academic Search

The chromosomal locations of restriction fragments of nuclear DNA that were homologous to four mitochondrial plasmid-like DNAs, namely, B1, B2, B3 and B4, were analyzed by restriction fragment length polymorphism (RFLP) analysis in cultivated rice. Nine kinds of fragments homologous to plasmidlike DNAs were analyzed for their segregation in three different F2 populations derived from intercrosses between rice subspecies; these

A. Kanazawa; N. Kishimoto; W. Sakamoto; R. Ohsawa; Y. Ukai; N. Tsutsumi; A. Hirai; A. Saito

1993-01-01

63

Differentiation of a specific Trichoderma biological control agent by restriction fragment length polymorphism (RFLP) analysis  

Microsoft Academic Search

A single biological control isolate belonging to the species aggregate Trichoderma harzianum Rifai was differentiated from other closely related Trichoderma species and commonly isolated mycoflora by molecular assay. Restriction fragment length polymorphism (RFLP) analysis enabled the differentiation of the single isolate; a hybridising band of 1.1 kb being diagnostic. Cross?hybridisation occurred against the DNA from 12 Trichoderma isolates, representative of

J. K. Bowen; S. C. Franicevic; R. N. Crowhurst; M. D. Templeton; A. Stewart

1996-01-01

64

Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis  

EPA Science Inventory

A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

65

Sizing of DNA fragments by flow cytometry  

SciTech Connect

Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1,29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis. We have demonstrated sizing of individual, stained DNA fragments with resolution approaching that of gel electrophoresis for moderately large fragments, but with significant reductions in the analysis time and the amount of sample required. Furthermore, system response is linear with DNA fragment length, in contrast to the logarithmic response in gel electrophoresis. There exists the potential to perform this sizing using relatively simple instrumentation, i.e. a continuous wave laser of low power and current mode detection.

Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Jett, J.H.; Keller, R.A.

1993-02-01

66

Sizing of DNA fragments by flow cytometry  

SciTech Connect

Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1,29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis. We have demonstrated sizing of individual, stained DNA fragments with resolution approaching that of gel electrophoresis for moderately large fragments, but with significant reductions in the analysis time and the amount of sample required. Furthermore, system response is linear with DNA fragment length, in contrast to the logarithmic response in gel electrophoresis. There exists the potential to perform this sizing using relatively simple instrumentation, i.e. a continuous wave laser of low power and current mode detection.

Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Jett, J.H.; Keller, R.A.

1993-01-01

67

A new MspI restriction fragment length polymorphism in the hemophilia B locus  

Microsoft Academic Search

Using a partial cDNA probe for human coagulation factor IX, we have detected a new restriction fragment length polymorphism in human DNA digested with MspI. The frequency of the minor allele is 0.20±0.05 and average heterozygosity is about 0.32. The MspI RELP is in strong linkage disequilibrium with the TaqI RFLP previously described, but should nevertheless be useful in segregation

G. Camerino; I. Oberlé; D. Drayna; J. L. Mandel

1985-01-01

68

Restriction Fragment Length Polymorphisms in the Mushrooms Agaricus brunnescens and Agaricus bitorquis  

PubMed Central

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species), were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates of both species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in Southern DNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wild type, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species. There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescens strains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA from heterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A. brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplast regenerates from one commercial strain and two strains collected in the wild. Images

Castle, Alan J.; Horgen, Paul A.; Anderson, James B.

1987-01-01

69

Restriction Fragment Length Polymorphism Linkage Map of Arabidopsis thaliana  

Microsoft Academic Search

We have constructed a restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the small flowering plant Arabidopsis thaliana. The map is based on the meiotic segregation of both RFLP and morphological genetic markers from five independent crosses. The morphological rnarkers on each of the five chromosomes were included in the crosses to allow alignment of the

Hong-Gil Nam; Francis Moonan; William D. B. Loos; Brian M. Hauge; Howard M. Goodman

1989-01-01

70

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding  

Microsoft Academic Search

Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability.

Tim Helentjaris; Gretchen King; Mary Slocum; Chris Siedenstrang; Sharon Wegman

1985-01-01

71

Mitochondrial restriction fragment length polymorphisms in wild Phaseolus vulgaris L.: insights on the domestication of the common bean  

Microsoft Academic Search

Previous examination of intraspecific mitochondrial DNA (mtDNA) diversity in common bean, Phaseolus vulgaris, showed that five restriction fragment length polymorphisms (RFLPs) distinguish the mitochondrial genomes of the two major gene pools of cultivated beans, the Mesoamerican and the Andean. In the study presented here, mtDNA was used to compare the amount of diversity in cultivated beans to that in collections

M. M. Khairallah; B. B. Sears; M. W. Adams

1992-01-01

72

Fragmentation of Genomic DNA using Microwave Irradiation  

PubMed Central

An unconventional approach for DNA fragmentation was investigated to explore its feasibility as an alternative to the existing DNA fragmentation techniques for next-generation DNA sequencing application. Current methods are based on strong-force liquid shearing or specialized enzymatic treatments. There are shortcomings for these platforms yet to be addressed, including aerosolization of genomic materials, which may result in the cross-contamination and biohazards; the difficulty in multiplexing; and the potential sequence biases. In this proof-of-concept study, we investigated the microwave irradiation as a simple, unbiased, and easy-to-multiplex way to fragment genomic DNA randomly. In addition, heating DNA at high temperature was attempted for the same purpose and for comparison. Adaptive focused acoustic sonication was used as the control. The yield and functionality for the DNA fragments and DNA fragment libraries were analyzed to assess the feasibility and use of the proposed approach. Both microwave irradiation and thermal heating can fragment genomic DNA to the size ranges suitable for next-generation sequencing (NGS) shotgun library preparation. However, both treatments caused severe reduction in PCR amplification efficiency, which led to low production in emulsion PCR (emPCR). The result was improved by amplification prior to emPCR. Further improvements, such as DNA strand repairing, are needed for the method to be applied practically in NGS.

Yang, Yu; Hang, Jun

2013-01-01

73

Restriction fragment length polymorphism species-specific patterns in the identification of white truffles.  

PubMed

A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations. PMID:9682488

Bertini, L; Potenza, L; Zambonelli, A; Amicucci, A; Stocchi, V

1998-07-15

74

The use of restriction fragment length polymorphisms in paternity analysis.  

PubMed Central

This paper examines the utility of restriction fragment length polymorphisms (RFLPs) for paternity analysis. While, on the average, 99% of falsely accused males can be excluded with the standard battery of blood group antigens, red cell enzymes, serum proteins, and HLA antigens, there are still mother-child pairs for whom the exclusion probability is not high. It has been suggested that additional resolution would be available with RFLPs. We have examined the strategic aspects of using RFLPs for paternity analysis, comparing the efficacy and cost of a multimarker haplotypic set with those of a comparable set of unlinked RFLPs, using published frequencies for the beta-globin complex, the serum albumin region, and the growth hormone region. There are four major findings. (1) Greater resolution is obtained with a carefully chosen set of tightly linked RFLPs producing chromosomal haplotypes than with a comparable set (same allele frequencies) of unlinked markers, but only if it is possible to establish linkage phase unambiguously. (2) Assay of linked sets is cheaper than is the assay of unlinked markers, but the cost advantage is optimized with sets of no more than two or three linked markers. (3) Also, with more than two or three tightly linked markers, the haplotypic frequencies are too poorly estimated to provide a reliable measure of the probability of paternity for unexcluded males, given the sample sizes likely to be available in the near future. (4) Optimal resolution, minimal cost, and acceptable accuracy are obtained with several independent sets of no more than two or three tightly linked RFLP markers each. With current technology, RFLP analysis is more expensive for the same level of genetic resolution than is the standard battery, but gradual replacement of the latter can be anticipated as economies of scale reduce the cost of the DNA technology.

Smouse, P E; Chakraborty, R

1986-01-01

75

Candida albicans- and Candida stellatoidea-specific DNA fragment.  

PubMed

DNA was isolated from whole cells of Candida albicans and digested with MspI restriction enzyme. In addition to the expected large number of low-molecular-weight DNA pieces resulting from the digestion, multiple high-molecular-weight (greater than 3.0 kilobase pairs) fragments were generated by this enzyme, which cleaves DNA at CCGG sequences. Some of these fragments appeared highly repeated. An MspI fragment which was similar in size to one of the repeat elements (2.9 kilobase pairs) was cloned into the ClaI site of the plasmid vector pBR322 and replicated in a suitable Escherichia coli host strain. The candidal fragment was radiolabeled and used to probe Southern blots of DNA from several Candida species, various other fungi, and mouse and human cells. Only DNA from C. albicans and a strain of Candida stellatoidea was found to contain sequences of significant homology for hybridization. The cloned fragment may possibly be of use as a DNA probe for detection of the presence of C. albicans. PMID:2460494

Cutler, J E; Glee, P M; Horn, H L

1988-09-01

76

In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization  

PubMed Central

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

Jiang, Xinglin; Yang, Jianming; Zhang, Haibo; Zou, Huibin; Wang, Cong; Xian, Mo

2012-01-01

77

Restriction enzyme mapping the ribosomal RNA genes in Solanum tuberosum: Potato rDNA restriction enzyme map  

Microsoft Academic Search

A restriction enzyme map of the ribosomal RNA genes (rDNA) in Solanum tuberosum cultivars Golden Wonder and Desiree has been constructed. An heterologous probe pTa 71 containing the rDNA derived from wheat was used to detect and map the corresponding region in potato genomic DNA fragments. rDNA repeats of cultivars Desiree and Golden Wonder are similar with respect to their

K. Harding

1991-01-01

78

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene, Hox-2.1  

Microsoft Academic Search

Restriction endonuclease fragment length polymorphisms (RFLPs) were found using the cDNA probe Hox-2.1 for the homeo box-2.1 gene in the mouse. Polymorphism was detected in restriction patterns generated by fragments fromHindIII digestion. The great majority of laboratory strains of mice carries theHox-2.1a allele. Only two laboratory strains carry theHox-2.1b allele. Among strains of wild origin, the European subspecies (Mus m.

Tomomasa Watanabe; Shigeo Masaki; Naoki Takahashi; Masahiko Nishimura; Hideki Kato

1988-01-01

79

A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci  

Microsoft Academic Search

Summary A detailed linkage map ofB. rapa (syn.campestris) was constructed based on segregation of 280 restriction fragment length polymorphism loci, detected by using 188 genomic DNA clones as probes on DNAs from a F2 population of Chinese cabbage ‘MichihilF’בSpring broccoli.’ These genetic markers covered 1,850 centiMorgans (cM) and defined ten linkage groups, which equals the haploid chromosome number of this

K. M. Song; J. Y. Suzuki; M. K. Slocum; P. M. Williams; T. C. Osborn

1991-01-01

80

Apoptotic DNA fragmentation and tissue homeostasis  

Microsoft Academic Search

DNA fragmentation is a hallmark of apoptosis. The tightly controlled activation of the apoptosis-specific endonucleases provides an effective means to ensure the removal of unwanted DNA and the timely completion of apoptosis. Over the past several years, crucial progress has been made in identifying the long-awaited apoptotic endonucleases, and their importance in tissue homeostasis is beginning to unfold. Here, we

Jianhua Zhang; Ming Xu

2002-01-01

81

Applications of restriction endonuclease fingerprinting of chromosomal DNA of Neisseria meningitidis.  

PubMed Central

Restriction endonucleases are bacterial enzymes that cleave DNA at specific sites. The resulting DNA fragments may be separated electrophoretically in gel to form specific restriction patterns. In the present study, the restriction endonuclease method was successfully adapted to the analysis of the chromosomal DNA of Neisseria meningitidis. The endonucleases HindIII and EcoRI provided optimal restriction patterns of ca. 50 well-separated lines. The pattern of each bacterial isolate was characteristic, stable, and reproducible. Despite some general similarity, the restriction patterns of the closely related B15 meningococci were surprisingly heterogeneous. Images

Bjorvatn, B; Lund, V; Kristiansen, B E; Korsnes, L; Spanne, O; Lindqvist, B

1984-01-01

82

RESTseq--efficient benchtop population genomics with RESTriction Fragment SEQuencing.  

PubMed

We present RESTseq, an improved approach for a cost efficient, highly flexible and repeatable enrichment of DNA fragments from digested genomic DNA using Next Generation Sequencing platforms including small scale Personal Genome sequencers. Easy adjustments make it suitable for a wide range of studies requiring SNP detection or SNP genotyping from fine-scale linkage mapping to population genomics and population genetics also in non-model organisms. We demonstrate the validity of our approach by comparing two honeybee and several stingless bee samples. PMID:23691128

Stolle, Eckart; Moritz, Robin F A

2013-05-17

83

RESTseq - Efficient Benchtop Population Genomics with RESTriction Fragment SEQuencing  

PubMed Central

We present RESTseq, an improved approach for a cost efficient, highly flexible and repeatable enrichment of DNA fragments from digested genomic DNA using Next Generation Sequencing platforms including small scale Personal Genome sequencers. Easy adjustments make it suitable for a wide range of studies requiring SNP detection or SNP genotyping from fine-scale linkage mapping to population genomics and population genetics also in non-model organisms. We demonstrate the validity of our approach by comparing two honeybee and several stingless bee samples.

Stolle, Eckart; Moritz, Robin F. A.

2013-01-01

84

Restriction endonucleases digesting DNA in PCR buffer  

Microsoft Academic Search

Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability\\u000a to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement\\u000a for additional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated\\u000a lambda DNA

Liu Xue-dong; Zheng Dong; Zhou Yan-na; Mao Wei-wei; Ma Jian-zhang

2005-01-01

85

Population Structure of the Atlantic Bottlenose Dolphin as Determined by Restriction Endonuclease Analysis of Mitochondrial DNA (Revised Version).  

National Technical Information Service (NTIS)

Restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) were used to test the discreteness of bottlenose dolphin (Tursiops truncatus) populations, with special emphasis on the Atlantic Ocean and Gulf of Mexico. Atlantic and Pacific dolphin ...

T. E. Dowling W. M. Brown

1992-01-01

86

Acclimatization of Pink Salmon Oncorhynchus gorbuscha Walbaum in the European North: mtDNA Restriction Data  

Microsoft Academic Search

Pink salmon spawners introduced into the White Sea basin (the Umba River) were compared to the spawners from the basin of the Sea of Okhotsk (the Ola River) using restriction analysis of two fragments of mitochondrial DNA (mtDNA). One of the fragments included genes ND5\\/ND6, the other, the cytochrome b gene and the D-loop. It was found that mtDNA variation

N. V. Gordeeva; E. A. Salmenkova; Yu. P. Altukhov

2004-01-01

87

Random DNA fragmentation with endonuclease V: application to DNA shuffling.  

PubMed

The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes. PMID:12490730

Miyazaki, Kentaro

2002-12-15

88

Restriction Fragment Length Polymorphism Linkage Map of Arabidopsis thaliana.  

PubMed Central

We have constructed a restriction fragment length polymorphism (RFLP) linkage map of the nuclear genome of the small flowering plant Arabidopsis thaliana. The map is based on the meiotic segregation of both RFLP and morphological genetic markers from five independent crosses. The morphological markers on each of the five chromosomes were included in the crosses to allow alignment of the RFLP map with the established genetic map. The map contains 94 new randomly distributed molecular markers (nine identified cloned Arabidopsis genes and 85 genomic cosmid clones) that detect polymorphisms between the Landsberg erecta and Columbia races. In addition, 17 markers from an independently constructed RFLP map of the Arabidopsis genome [Chang, C., Bowman, J.L., DeJohn, A.W., Lander, E.S., and Meyerowitz, E.M. (1988). Proc. Natl. Acad. Sci. USA 85, 6856-6860] have been included to permit integration of the two RFLP maps.

Nam, H. G.; Giraudat, J.; Den Boer, B.; Moonan, F.; Loos, WDB.; Hauge, B. M.; Goodman, H. M.

1989-01-01

89

Polimorfismo dos Fragmentos de Restricao do DNA Ribossomal em Taxonomia Molecular de Leveduras: Estudo de um Genero (Restriction Fragment Polymorphism of Ribosomal DNA in Yeasts Molecular Taxonomy: Study of a Genus).  

National Technical Information Service (NTIS)

The study explored the possibility of obtaining species-specific probes based on cloned spacer sequences of rDNA using the type strain of the yeast Metschnikowia reukaufii as a model. The study isolated, cloned and mapped its rDNA. Subsequently sub-clones...

M. O. I. Henriques

1990-01-01

90

Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens  

Microsoft Academic Search

For restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the rDNA fragments of 1.5 kb were amplified by polymerase chain reaction (PCR) from crude cell lysates of various methanogenic species which were prepared by a combined technique of ultrasonic treatment and protease digestion. The PCR products were purified by the polyethylene glycol precipitation method and treated with various

Akira Hiraishi; Yoichi Kamagata; Kazunori Nakamura

1995-01-01

91

Diffusion of the Restriction Nuclease EcoRI along DNA  

PubMed Central

Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring 1-dimensional diffusion along DNA based on the ratio of the dissociation rates of EcoRI from DNA fragments containing one and two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure indicating the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 × 104 bp2sec?1. EcoRI is able to diffuse ~150 base pairs on average along DNA in 1 second. This diffusion rate is about 2000-fold slower than the diffusion of the free protein in solution. A factor of 40–50 can be accounted for by a rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for remaining activation energy: the salt bridges between the DNA and protein are transiently broken or the water structure at the protein-DNA interface is disrupted as the two surfaces move past one another.

Sidorova, Nina Y

2009-01-01

92

Restriction fragment length polymorphism analysis of loci associated with disease resistance genes and developmental traits in Pisum sativum L  

Microsoft Academic Search

An F2 population of pea (Pisum sativum L.) consisting of 174 plants was analysed by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. Ascochyta pisi race C resistance, plant height, flowering earliness and number of nodes were measured in order to map the genes responsible for their variation. We have constructed a partial linkage map including

E. Dirlewanger; P. G. Isaac; S. Ranade; M. Belajouza; R. Cousin; D. Vienne

1994-01-01

93

Taenia saginata: Differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay  

Microsoft Academic Search

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected

Cáris Maroni Nunes; Ana Karina Kerche Dias; Francisca Elda Ferreira Dias; Sérgio Moraes Aoki; Henrique Borges de Paula; Luis Gustavo Ferraz Lima; José Fernando Garcia

2005-01-01

94

Sensitive PCR-Restriction Fragment Length Polymorphism Assay for Detection and Genotyping of Giardia duodenalis in Human Feces  

Microsoft Academic Search

An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence- stained fecal

C. F. L. Amar; P. H. Dear; S. Pedraza-Diaz; N. Looker; E. Linnane; J. McLauchlin

2002-01-01

95

Regional localization of chromosome 3-specific DNA fragments by using a hybrid cell deletion mapping panel.  

PubMed Central

A series of human chromosome 3-specific DNA fragments isolated and characterized from a lamda phage genomic library were regionally localized on human chromosome 3. This was accomplished using filter hybridization blot analysis of a human chromosome 3 hybrid cell deletion mapping panel. Twenty-three new anonymous DNA fragments were assigned to one of four physical regions of chromosome 3. Seventeen DNA fragments were mapped to the long arm of chromosome 3, including one DNA fragment that demonstrated a restriction fragment length polymorphism (RFLP). Five DNA fragments were assigned to 3p14.2----pter, including one highly polymorphic fragment sublocalized at 3p25----pter by in situ hybridization. This DNA fragment is the second reported distal 3p polymorphic probe. One DNA fragment was localized to 3p14----p14.2. In addition, three fragments previously assigned to chromosome 3 were confirmed. Polymorphic DNA probes DNF15S2 (formerly D1S1) and D3S2 were mapped to 3p14.2----pter. The previous 3p25 in situ localization of the c-raf-1 oncogene was supported by deletion panel mapping. The physical localization of these twenty-three new DNA fragments has more than doubled the number of cloned DNA fragments assigned to chromosome 3. These and future regional assignments of DNA fragment probes will facilitate construction of both a physical and genetic linkage map of chromosome 3. They may also be useful in characterizing the chromosomal and molecular aberrations involved in small-cell lung cancer (SCLC), renal cell carcinoma, other malignancies, and the 3p14.2 common fragile site. Images p[446]-a Figure 2 Figure 3

Gerber, M J; Drabkin, H A; Firnhaber, C; Miller, Y E; Scoggin, C H; Smith, D I

1988-01-01

96

Restriction Endonuclease Analysis of Marek's Disease Virus DNA and Homology between Strains  

Microsoft Academic Search

SUMMARY The restriction endonuclease patterns of viral DNA obtained from serotypes 1 and 2 strains of Marek's disease virus have been compared and homology between the strains examined by hybridization. The results have shown that HPRS 16 (serotype 1) DNA has a structure similar to its attenuated variant HPRS 16\\/att except for a few fragments that are present only in

L. J. N. Ross; B. Milne; P. M. Biggs

1983-01-01

97

Repair of base alkylation damage in targeted restriction endonuclease sequences of plasmid DNA  

Microsoft Academic Search

Sequence specific ethylation damage and repair of ethyl-adducts in selected restriction endonuclease recognition sites withinp220-ras plasmid DNA was assessed by a modified Southern blotting coupled immunoprobing technique. In situ UV irradiation of DNA in gels clearly ameliorated the immunodetection of minute amounts of facultative fragments generated due to inhibition of enzyme cleavage site by covalent alkylation modification of the cognate

Javed Musarrat; Jasna Arezina-Wilson; Altaf A. Wani

1995-01-01

98

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene, Hox-2.1  

Microsoft Academic Search

Restriction endonuclease fragment length polymorphisms (RFLPs) were found using the cDNA probe Hox-2.1 for the homeo box-2.1\\u000a gene in the mouse. Polymorphism was detected in restriction patterns generated by fragments fromHindIII digestion. The great majority of laboratory strains of mice carries theHox-2.1\\u000a a allele. Only two laboratory strains carry theHox-2.1\\u000a b allele. Among strains of wild origin, the European subspecies

Tomomasa Watanabe; Shigeo Masaki; Naoki Takahashi; Masahiko Nishimura; Hideki Kato

1988-01-01

99

Automated DNA fragments recognition and sizing through AFM image processing  

Microsoft Academic Search

This paper presents an automated algorithm to determine DNA fragment size from atomic force microscope images and to extract the molecular profiles. The sizing of DNA fragments is a widely used procedure for investigating the physical properties of individual or protein-bound DNA molecules. Several atomic force microscope (AFM) real and computer-generated images were tested for different pixel and fragment sizes

Elisa Ficarra; Luca Benini; Enrico Macii; Giampaolo Zuccheri

2005-01-01

100

Molecular marker analysis of Helianthus annuus L. 1. Restriction fragment length polymorphism between inbred lines of cultivated sunflower  

Microsoft Academic Search

cDNA and PstI genomic clones have been used to assess levels of restriction fragment length polymorphism (RFLP) in Helianthus annuus and to determine the inter-relationships between a diverse set of 24 inbred lines. Of the cDNA clones screened 45% were useful as RFLP probes, compared to less than 20% from the PstI library, which showed high levels of redundancy for

S. T. Berry; R. J. Allen; S. R. Barnes; P. D. S. Caligari

1994-01-01

101

Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter.  

PubMed

 DNA manipulations using a completely chemistry-based DNA cutter (ARCUT) have been reviewed. This cutter, recently developed by the authors, is composed of Ce(IV)/EDTA complex and two strands of pseudo-complementary peptide nucleic acid. The site-selective scission proceeds via hydrolysis of targeted phosphodiester linkages, so that the resultant scission fragments can be easily ligated with other fragments by using DNA ligase. Importantly, scission-site and site-specificity of the cutter are freely tuned in terms of the Watson-Crick rule. Thus, when one should like to manipulate DNA according to the need, he or she does not have to think about (1) whether appropriate "restriction enzyme sites" exist near the manipulation site and (2) whether the site-specificity of the restriction enzymes, if any, are sufficient to cut only the aimed position without chopping the DNA at non-targeted sites. Even the human genome can be manipulated, since ARCUT can cut the genome at only one predetermined site. Furthermore, the cutter is useful to promote homologous recombination in human cells, converting a site to desired sequence. The ARCUT-based DNA manipulation should be promising for versatile applications. PMID:23385238

Komiyama, Makoto

2013-02-05

102

Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure  

Microsoft Academic Search

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68

Markus Egert; Michael W. Friedrich

2003-01-01

103

Restriction analysis of specific DNA sequences of five homothallic species of Neurospora  

SciTech Connect

Nuclear DNA's from five homothallic species of Neurospora: N. africana, N. dodgei, N. lineolata, N. galapagosensis and N. terricola were isolated and characterized. From these total nuclear DNA's, specific DNA sequences were isolated by CsCl buoyant density gradient equilibrium centrifugation. These selected DNA's were restricted separately with EcoR1, Bam H1, Hind III, XBa 1 and Hinc II. DNA digests were electrophorused in varying agarose gel concentration (0.7%, 0.8% and 1%) and the sizes of fragments generated with these endonucleases were estimated against lambda phage DNA and pMF2 DNA fragments. EcoR1 generated four DNA size variants of about 20.0 kb, 7.5 kb, 5.5 b and 5.23 kb in N. terricola except the 10.5 kb fragment. Instead, N. lineolata showed three distinct DNA size variants of about 15.0 kb, 12.0 kb and 8.2 kb with Bam H1 treatment. Similarly we have noticed unique DNA restriction bands when treated with a few other restriction enzymes like Xba 1 and Hind III. These results suggest distinct rDNA nucleotide sequence differences among homothallic species of Neurospora studied.

Attoh, G.; Dutta, S.K.

1983-01-01

104

Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA.  

PubMed

In traditional electrophoresis mobility shift assay (EMSA) a single (32)P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. An extension of this method uses a population of DNA restriction fragments derived from long genomic regions for the identification of fragments containing protein binding regions. Although the method allows simultaneous analysis of large fragments, it is relatively laborious and can be used to detect only fragments containing high affinity protein binding sites. Here we describe an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. With this strategy restriction fragments, derived from genomic DNA (<10 kb), containing high as well as low affinity protein binding regions may be easily identified. PMID:23436361

Kaer, Kristel; Speek, Mart

2013-01-01

105

Anatomy of Herpes Simplex Virus DNA: Strain Differences and Heterogeneity in the Locations of Restriction Endonuclease Cleavage Sites  

Microsoft Academic Search

Digestion of herpes simplex virus DNA by the HinIII or EcoRI restriction endonucleases yielded 11 to 15 fragments with molecular weights between 1 × 106 and 28 × 106. The electrophoretic profiles obtained in 0.3% agarose gels with DNA fragments from nine different strains of herpes simplex virus type 1 could be readily differentiated from the patterns exhibited by the

G. S. Hayward; N. Frenkel; B. Roizman

1975-01-01

106

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.  

PubMed Central

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb. Images

Morimoto, R; Lewin, A; Rabinowitz, M

1977-01-01

107

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.  

PubMed

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb. PMID:333388

Morimoto, R; Lewin, A; Rabinowitz, M

1977-07-01

108

Use of restriction fragment length polymorphisms to investigate strain variation within Neisseria meningitidis  

SciTech Connect

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P{sup 32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population.

Williams, S.D.

1989-01-01

109

Use of Restriction Fragment Length Polymorphisms to Investigate Strain Variation Within Neisseria Meningitidis.  

NASA Astrophysics Data System (ADS)

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty -six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P ^{32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analysed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population. This analysis demonstrates the lack of structure within Neisseria meningitidis due primarily to a heterogenous population and the lack of geographic segregation. The potential utility of this technique as a tool in epidemiologic surveillance is addressed. Further work is needed in the evaluation of RFLP analysis in the taxonomy bacteria.

Williams, Shelley Diane

110

COMPARISON OF THE RESTRICTION ENDONUCLEASE DIGESTION PATTERNS OF MITOCHONDRIAL DNA FROM NORMAL AND MALE STERILE CYTOPLASMS OF ZEA MAYS L  

Microsoft Academic Search

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, Sal1 and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial

KATHLEEN S. BORCK; VIRGINIA WALBOT

111

Genomic variation of aquatic birnaviruses analyzed with restriction fragment length polymorphisms.  

PubMed Central

Aquatic birnaviruses are the most ubiquitous and diverse group of viruses in the family Birnaviridae. Several cause different diseases in a variety of fish species, such as infectious pancreatic necrosis virus in salmonids in North America, Europe, and Asia and European eel virus in eel in Asia. Most isolates are antigenically related and belong to a single serogroup (serogroup A) comprising nine serotypes. Previous studies with monoclonal antibodies have demonstrated considerable variation in epitope profiles even among strains within a single serotype. The few studies of genomic variation among these viruses, which have focused on the NS/VP3 coding region, demonstrated the existence of several genogroups that generally did not correlate with antigenic groups. In this study, PCR was used to amplify a 1,180-bp cDNA genomic fragment representing most of the VP2 (the major outer capsid protein) coding region from five serotype A type strains and 17 Asian isolates. The PCR products were digested with nine different restriction enzymes. Restriction fragment length polymorphism profiles demonstrated heterogeneity among the tested viruses; however, the isolates from Asia were closely related to each other. Cluster analysis of the restriction fragment length polymorphism patterns demonstrated that these viruses could be divided into four major genogroups. In contrast to previous studies of variation in the NS/VP3 coding region, these genogroups based on variation in the VP2 coding region correlated with a serological classification based on VP2-specific monoclonal antibody reaction patterns. Furthermore, all Asian isolates tested belonged to one genogroup typified by the serotype type strain Ab.

Lee, M K; Blake, S L; Singer, J T; Nicholson, B L

1996-01-01

112

Identification of cagA tyrosine phosphorylation DNA motifs in Helicobacter pylori isolates from peptic ulcer patients by novel PCR-restriction fragment length polymorphism and real-time fluorescence PCR assays.  

PubMed

Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation. PMID:12843050

Owen, Robert J; Sharp, Sally I; Chisholm, Stephanie A; Rijpkema, Sjoerd

2003-07-01

113

Identification of cagA Tyrosine Phosphorylation DNA Motifs in Helicobacter pylori Isolates from Peptic Ulcer Patients by Novel PCR-Restriction Fragment Length Polymorphism and Real-Time Fluorescence PCR Assays  

PubMed Central

Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation.

Owen, Robert J.; Sharp, Sally I.; Chisholm, Stephanie A.; Rijpkema, Sjoerd

2003-01-01

114

A 'dirty' business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi.  

PubMed

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. 'extra peaks') per PCR primer-restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description. PMID:16499709

Avis, Peter G; Dickie, Ian A; Mueller, Gregory M

2006-03-01

115

DNA fragmentation in chicken spermatozoa during cryopreservation.  

PubMed

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at -196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation. PMID:21396690

Gliozzi, T M; Zaniboni, L; Cerolini, S

2011-03-11

116

Comparison of the Restriction Endonuclease Digestion Patterns of Mitochondrial DNA from Normal and Male Sterile Cytoplasms of ZEA MAYS L.  

PubMed

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, SalI and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization behavior.-The total SalI digest and the larger PstI fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize. PMID:17246091

Borck, K S; Walbot, V

1982-09-01

117

Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities.  

PubMed

DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However, these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis. This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities. PMID:21274516

Wang, Tingting; Zhang, Xiaojun; Zhang, Menghui; Wang, Linghua; Zhao, Liping

2011-01-28

118

Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA  

Microsoft Academic Search

BACKGROUND: In a traditional electrophoresis mobility shift assay (EMSA) a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments

Kristel Kaer; Kert Mätlik; Madis Metsis

2008-01-01

119

Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis.  

PubMed

Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundaçăo de Medicina Tropical do Amazonas--FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods. PMID:20640387

Santos, M S; Souza, E S; S Junior, R M; Talhari, S; Souza, J V B

2010-07-16

120

Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis.  

PubMed Central

Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis of M. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in different countries. Images

Skuce, R A; Brittain, D; Hughes, M S; Beck, L A; Neill, S D

1994-01-01

121

A simple restriction fragment PCR approach for discrimination of humanpathogenic Old World animal Orthopoxvirus species.  

PubMed

There are reliable polymerase chain reaction assays available for exclusion of Variola virus from other poxviruses. However, the discrimination of humanpathogenic animal Orthopoxviridae is more challenging because of the high genomic conservation. Based on the variability of the A36R gene, we describe a simple 20 min PCR assay followed by a 1 h digest with 3 different restriction enzymes. This assay enables rapid discrimination between Cowpox virus and Monkeypox virus and discrimination of the most prevalent members of the Vaccinia virus and Camelpox virus. The test was orthopoxvirus specificand did not react with parapox (Orf) virus. Moreover, the amplified fragments were also well suited for additional genotyping by direct DNA sequencing. PMID:18388986

Huemer, Hartwig P; Hönlinger, Bettina; Höpfl, Reinhard

2008-02-01

122

High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis  

Microsoft Academic Search

We have developed an automated, high-throughput fingerprinting technique for large genomic DNA fragments suitable for the construction of physical maps of large genomes. In the technique described here, BAC DNA is isolated in a 96-well plate format and simultaneously digested with four 6-bp-recognizing restriction endonucleases that generate 3? recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end.

Ming-Cheng Luo; Carolyn Thomas; Frank M You; Joseph Hsiao; Shu Ouyang; C. Robin Buell; Marc Malandro; Patrick E McGuire; Olin D Anderson; Jan Dvorak

2003-01-01

123

Terminal Restriction Fragment Length Polymorphism Analysis Program, a Web-Based Research Tool for Microbial Community Analysis  

PubMed Central

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses.html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5? terminus of the user-specified primer to the 3? terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.

Marsh, Terence L.; Saxman, Paul; Cole, James; Tiedje, James

2000-01-01

124

Effect of aging and dietary restriction on DNA repair  

SciTech Connect

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.

Weraarchakul, N.; Strong, R.; Wood, W.G.; Richardson, A.

1989-03-01

125

Genotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction  

SciTech Connect

Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantification of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. The authors describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 10{sup 8} copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.

Sandy, M.S.; Chiocca, S.M.; Cerutti, P.A. (Swiss Inst. for Experimental Cancer Research, Lausanne (Switzerland))

1992-02-01

126

Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes  

Microsoft Academic Search

Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56,denA anddenB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA withEcoRI have been cloned using the vector plasmid pCR1.

Tom Mattson; Griet Van Houwe; Antoinette Bolle; Gerald Selzer; Richard Epstein

1977-01-01

127

A study of restriction fragment length polymorphisms at the human alpha-1-antitrypsin locus  

Microsoft Academic Search

A cloned cDNA for a-1-antitrypsin (a-1-AT) was selected from a human liver cDNA library. The identity of the clone was established by hybrid-selected translation and partial DNA sequencing. The cDNA was used as a probe to search for restriction site polymorphisms (RSPs) near the a-1-AT gene. Only two RSPs were found using 29 different restriction enzymes. Each of these polymorphisms

K. J. Matteson; H. Ostrer; A. Chakravarti; K. H. Buetow; W. E. O'Brien; A. L. Beaudet; J. A. Phillips

1985-01-01

128

Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis.  

PubMed Central

Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds. The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds. Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis. The procedures, which do not require any special techniques, are described in detail. The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm. Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E. coli DNA and calf thymus DNA are given. Images

Muller, W; Hattesohl, I; Schuetz, H J; Meyer, G

1981-01-01

129

GP7 induces internucleosomal DNA fragmentation independent of caspase activation and DNA fragmentation factor in NB4 cells.  

PubMed

DNA fragmentation into internucleosomal fragments is the best recognized biochemical event of apoptosis. Two major caspase pathways have been identified in the signal transduction leading to DNA fragmentation: the receptor pathway and the mitochondrial pathway. DNA fragmentation factor (DFF) has been identified as a major apoptotic endonuclease in the internucleosomal DNA fragmentation process. However, the potential roles of caspases and DFF in internucleosomal DNA fragmentation induced by specific stimuli still need to be investigated since caspase-independent pathways and nuclease(s) other than DFF also play important roles during this process. In the present study, we investigated the activity of GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university, to induce apoptosis of the human leukemia cell line NB4. GP7 induced the release of cytochrome-c from mitochondria, activations of caspase-3, -8, and -9, cleavage of DFF45/inhibitor of caspase-activated DNase, activation of DFF40/caspase-activated DNase, and apoptotic DNA fragmentation in NB4 cells. The broad-spectrum caspase inhibitor zVAD-fmk abrogated GP7-induced caspase-3, -8, and -9 activations but could not inhibit GP7-induced apoptotic DNA fragmentation in NB4 cells. Our findings suggest that GP7-induced apoptotic DNA fragmentation in NB4 cells is independent of caspase activation and DFF, although they are closely involved in this process. PMID:17549379

Qi, She-Ning; Jing, Yuan-Xue; Dong, Gen-Xi; Chen, Yan; Yoshida, Akira; Ueda, Takanori

2007-07-01

130

Genetic Identification of the Main Opportunistic Mucorales by PCR-Restriction Fragment Length Polymorphism  

PubMed Central

Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis.

Machouart, M.; Larche, J.; Burton, K.; Collomb, J.; Maurer, P.; Cintrat, A.; Biava, M. F.; Greciano, S.; Kuijpers, A. F. A.; Contet-Audonneau, N.; de Hoog, G. S.; Gerard, A.; Fortier, B.

2006-01-01

131

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.  

PubMed

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses. PMID:22038597

Andeer, Peter; Strand, Stuart E; Stahl, David A

2011-10-28

132

Protein fragment complementation in M.HhaI DNA methyltransferase  

Microsoft Academic Search

The 5mC DNA methyltransferase M.HhaI can be split into two individually inactive N- and C-terminal fragments that together can form an active enzyme in vivo capable of efficiently methylating DNA. This active fragment pair was identified by creating libraries of M.HhaI gene fragment pairs and then selecting for the pairs that code for an active 5mC methyltransferase. The site of

Wonchae Choe; Srinivasan Chandrasegaran; Marc Ostermeier

2005-01-01

133

THE USE OF RESTRICTION ENDONUCLEASES TO MEASURE MITOCHONDRIAL DNA SEQUENCE RELATEDNESS IN NATURAL POPULATIONS. I. POPULATION STRUCTURE AND EVOLUTION IN THE GENUS PEROMYSCUS  

Microsoft Academic Search

In this study we introduce to natural population analysis a molecular tech- nique that involves the use of restriction endonucleases to compare mitochon- drial DNA (mtDNA) sequences. We have examined the fragment patterns produced by six restriction endonucleases acting upon mtDNA isolated from 23 samples of three species of the rodent Peromyscus. Our observations confirm the following conclusions derived from

JOHN C. AVISE; ROBERT A. LANSMAN

134

A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma.  

PubMed Central

Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease. Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany. Southern analysis using a human progesterone receptor (hPR) cDNA probe identified a germline TaqI restriction fragment length polymorphism (RFLP) defined by two alleles: T1, represented by a 2.7 kb fragment; and T2, represented by a 1.9 kb fragment and characterised by an additional TaqI restriction site with respect to T1. An over-representation of T2 in ovarian cancer patients compared with controls in the pooled Irish/German population (P < 0.025) was observed. A difference (P < 0.02) in the distribution of the RFLP genotypes between Irish and German control populations was also observed. The allele distributions could not be shown to differ significantly from Hardy-Weinberg distribution in any subgroup. Using hPR cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene. Images Figure 2 Figure 3 Figure 4

McKenna, N. J.; Kieback, D. G.; Carney, D. N.; Fanning, M.; McLinden, J.; Headon, D. R.

1995-01-01

135

DNA Nucleotide Sequence Restricted by the RI Endonuclease  

Microsoft Academic Search

The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage lambda has been determined. The 5'-terminal nucleotide labeled with 32P and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in lambda DNA was

Joe Hedgpeth; Howard M. Goodman; Herbert W. Boyer

1972-01-01

136

Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine.  

PubMed Central

Phage fd RF I DNA1 about 90% substituted by deoxy-4-thiothymidine (s4Td) in the codogenic strand was synthesized by the simultaneous actions of DNA polymerase I and DNA ligase. While the rate of DNA synthesis was considerably reduced, the yield the rate of DNA synthesis was considerably reduced, the yield was not affected in the presence of s4TdTP. The conversion of RF II to RF I DNA by DNA ligase was even improved. This effect seems to be related with an altered ratio of affinity of polymerase and ligase for the s4Td-containing substrate. The presence of the base analogue in the DNA was verified independently by chromatographic and spectroscopic methods. The modified genome could be cleaved by restriction endonucleases Hpa II (C/CGG)d and Taq I (T/CGA)d. A number of the fragments produced showed altered mobilities under the conditions of polyacrylamide gel electrophoresis. Images

Hofer, B; Koster, H

1981-01-01

137

Analysis of the genomic termini of tupaia herpesvirus DNA by restriction mapping and nucleotide sequencing.  

PubMed Central

A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging. Images

Albrecht, M; Darai, G; Flugel, R M

1985-01-01

138

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.  

PubMed Central

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.

Liu, W T; Marsh, T L; Cheng, H; Forney, L J

1997-01-01

139

Examination of Genetic Relatedness of Marine Synechococcus spp. by Using Restriction Fragment Length Polymorphisms.  

PubMed

The relatedness of several marine Synechococcus spp. was estimated by DNA hybridization. Strains isolated from various geographical locations and representing a diversity of DNA base compositions and phycobiliprotein profiles were compared by restriction fragment length polymorphisms for a number of genes. DNAs from two marine red algae and a cryptomonad alga (which exhibit a phycobiliprotein composition similar to that of the marine Synechococcus spp.) and Synechococcus strain PCC6301 (Anacystis nidulans) were also included in the comparison. Strains WH8008, WH8018, and WH7805 were shown to be very similar to one another, as were strains WH7802 and WH7803. Strains WH8110 and WH5701 were clearly unrelated to any of the other strains, and no marine Synechococcus isolate showed any similarity to the freshwater Synechococcus strain PCC6301 or the eucaryotic algae. The method is relatively straightforward and sensitive and uses a variety of basic molecular biology techniques. Its utility in ascertaining the genetic relatedness and diversity of marine Synechococcus spp. and possible extension to field studies are discussed. PMID:16347797

Douglas, S E; Carr, N

1988-12-01

140

Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods  

PubMed Central

Background Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.

2013-01-01

141

Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.  

PubMed Central

A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found. Images

Mizuuchi, K; Nash, H A

1976-01-01

142

Heterothallic species of neurospora are distinguishable by restriction analysis of their nuclear rDNA sequences  

SciTech Connect

Restriction analysis of rDNAs was used to distinguish nuclear rDNA's of three different reference strains of heterothallic species of the genus Neurospora: N. crassa 74A (FGSC number987), N. intermedia P420 (FGSC number2316), and N. sitophila 10B (FGSC number580). Two approaches were adopted: (1) Nuclear DNA's of these three Neurospora species were treated with various restriction enzymes. Against the streaks of nuclear DNAs on the 0.7% agarose gels background bands were visible. These background bands are visible because rDNA sequences of Neurospora species exist in multiple copies within the nuclear DNA's. (2) The second approach was comparison of auto-radiographs of hybrid molecules of Southern blot transfers of restricted nuclear DNAs and /sup 32/P-labelled nick translated rDNA's (referred to as rDNA probe) isolated from N. crassa slime mutant (FGSC number1118), rDNA cloned into pBR322. A summary of restricted fragment sizes as seen in the gels and in autoradiographs of Southern blots of the respective gels is presented.

Chambers, C.; Dutta, S.K.

1983-01-01

143

Detection of Disease-Specific Restriction Fragment Length Polymorphisms in Pemphigus Vulgaris Linked to the DQw1 and DQw3 Alleles of the HLA-D Region  

Microsoft Academic Search

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQbeta , DQalpha , and DRbeta cDNA probes. Hybridization with the DQbeta probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate

Fanny Szafer; Chaim Brautbar; Eli Tzfoni; Gad Frankel; Lenny Sherman; Itzhak Cohen; Shoshana Hacham-Zadeh; Werner Aberer; Gerhard Tappeiner; Karl Holubar; Lawrence Steinman; Adam Friedmann

1987-01-01

144

Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities  

PubMed Central

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.

Kent, Angela D.; Smith, Dan J.; Benson, Barbara J.; Triplett, Eric W.

2003-01-01

145

FDA Information on Gardasil – Presence of DNA Fragments ...  

Center for Biologics Evaluation and Research (CBER)

The FDA has recently received inquiries regarding the presence of human papillomavirus (HPV) DNA fragments in Gardasil and is aware that ... More results from www.fda.gov/biologicsbloodvaccines/vaccines/approvedproducts

146

Rapid identification of filamentous actinomycetes to the genus level using genus-specific 16S rRNA gene restriction fragment patterns  

Microsoft Academic Search

A rapid method for identifying filamentous actinomycete genera was developed based on 16S rRNA gene restriction fragment patterns. The patterns were generated by using specific restriction endonucleases to perform in silico digestions on the 16S rRNA gene sequences of all validly published filamentous actinomycete species. The method was applied to identifying actinomycete isolates from soil. Amplified 16S rDNA of soil

Andrew E. Cook; Paul R. Meyers

2003-01-01

147

Automated sizing of DNA fragments in atomic force microscope images  

Microsoft Academic Search

Current techniques used to measure lengths of DNA fragments in atomic force microscope (AFM) images require a user to operate\\u000a interactive software and execute tedious error-prone cursor selections. An algorithm is proposed which provides an automated\\u000a method for determining DNA fragment lengths from AFM images without interaction from the computer operator (e.g. cursor selections\\u000a or mouse clicks). The approach utilises

T. S. Spisz; Y. Fang; R. H. Reeves; C. K. Seymour; I. N. Bankman; J. H. Hoh

1998-01-01

148

DNA fragmentation of spermatozoa and assisted reproduction technology  

Microsoft Academic Search

Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in

Ralf Henkel; Eva Kierspel; Marjam Hajimohammad; Thomas Stalf; Christiaan Hoogendijk; Claas Mehnert; Roelof Menkveld; Wolf-Bernhard Schill; Thinus F Kruger

2003-01-01

149

Restriction endonucleases functionally interacting with two DNA sites  

Microsoft Academic Search

Simultaneous interaction with two recognition sites was found to be a precondition for DNA cleavage by certain type-II and type-III restriction endonucleases. Nevertheless, the molecular mechanisms of the protein-DNA interaction are different between members of both classes of enzymes.

Detlev H. Krüger; Dagmar Kupper; Andreas Meisel; Mike Tierlich; Monika Reuter; Cornelia Schroeder

1995-01-01

150

Nonhomologous end joining during restriction enzyme-mediated DNA integration in Saccharomyces cerevisiae.  

PubMed

The BamHI restriction enzyme mediates integration of nonhomologous DNA into the Saccharomyces cerevisiae genome (R. H. Schiestl and T. D. Petes, Proc. Natl. Acad. Sci. USA 88:7585-7589, 1991). The present study investigates the mechanism of such events: in particular, the mediating activity of various restriction enzymes and the processing of resultant fragment ends. Our results show that in addition to BamHI, BglII and KpnI increase DNA integration efficiencies severalfold, while Asp718, HindIII, EcoRI, SalI, SmaI, HpaI, MscI, and SnaBI do not. Secondly, the three active enzymes stimulated integrations only of fragments containing 5' or 3' overhangs but not of blunt-ended fragments. Thirdly, integrations mediated by one enzyme and utilizing a substrate created by another required at least 2 bp of homology. Furthermore, an Asp718 fragment possessing a 5' overhang integrated into a KpnI (isoschizomer) site possessing a 3' overhang, most likely by filling of the 5' overhang followed by 5' exonuclease digestion to produce a 3' end. We classified and analyzed the restriction enzyme-mediated integration events in the context of their genomic positions. The majority of events integrated into single sites. In the remaining 6 of 19 cases each end of the plasmid inserted into a different sequence, producing rearrangements such as duplications, deletions, and translocations. PMID:9488490

Manivasakam, P; Schiestl, R H

1998-03-01

151

Short read DNA fragment anchoring algorithm  

PubMed Central

Background The emerging next-generation sequencing method based on PCR technology boosts genome sequencing speed considerably, the expense is also get decreased. It has been utilized to address a broad range of bioinformatics problems. Limited by reliable output sequence length of next-generation sequencing technologies, we are confined to study gene fragments with 30~50 bps in general and it is relatively shorter than traditional gene fragment length. Anchoring gene fragments in long reference sequence is an essential and prerequisite step for further assembly and analysis works. Due to the sheer number of fragments produced by next-generation sequencing technologies and the huge size of reference sequences, anchoring would rapidly becoming a computational bottleneck. Results and discussion We compared algorithm efficiency on BLAT, SOAP and EMBF. The efficiency is defined as the count of total output results divided by time consumed to retrieve them. The data show that our algorithm EMBF have 3~4 times efficiency advantage over SOAP, and at least 150 times over BLAT. Moreover, when the reference sequence size is increased, the efficiency of SOAP will get degraded as far as 30%, while EMBF have preferable increasing tendency. Conclusion In conclusion, we deem that EMBF is more suitable for short fragment anchoring problem where result completeness and accuracy is predominant and the reference sequences are relatively large.

Wang, Wendi; Zhang, Peiheng; Liu, Xinchun

2009-01-01

152

Chloroplast DNA variability in the genus Helianthus: restriction analysis and S1 nuclease mapping of DNA-DNA heteroduplexes  

Microsoft Academic Search

Chloroplast DNA (cpDNA) from 36 wild species of the genus Helianthus has been analysed with three restriction endonucleases (Bam HI, Hind III and Sst I). Out of the 71 restriction sites described on the reference cpDNA (sunflower cpDNA), three insertions\\/deletions and seven site modifications were detected during the survey of the other cpDNAs.

Pascale Serror; Françoise Heyraud; Philippe Heizmann

1990-01-01

153

Restriction Polymorphism of Mitochondrial DNA in Koreans and Mongolians  

Microsoft Academic Search

Using the data on mitochondrial DNA (mtDNA) restriction polymorphism, the gene pools of Koreans (N = 164) and Mongolians (N = 48) were characterized. It was demonstrated that the gene pools were represented by the common set of mtDNA haplogroups of East Asian origin (M*, M7, M8a, M10, C, D4, G*, G2, A, B*, B5, F1, and N*). In addition

M. V. Derenko; A. V. Lunkina; B. A. Malyarchuk; I. A. Zakharov; Ts. Tsedev; K. S. Park; Y. M. Cho; H. K. Lee; Ch. H. Chu

2004-01-01

154

Pig mitochondrial DNA: Polymorphism, restriction map orientation, and sequence data  

Microsoft Academic Search

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1 four-nucleotide-recognizing enzyme. Pigs including Taiwan native breeds and miniature strains maintained in Japan were examined in this study; four commercial breeds of pigs and Japanese wild boars have been investigated earlier [Watanabe, T., et al. (1985). Biochem. Genet.23:105]. mtDNA

Tomomasa Watanabe; Yukimasa Hayashi; Jun Kimura; Yukio Yasuda; Naruya Saitou; Takeshi Tomita; Nobuaki Ogasawara I

1986-01-01

155

Generation of a Restriction Fragment Length Polymorphism Linkage Map for Toxoplasma Gondii  

PubMed Central

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.

Sibley, L. D.; LeBlanc, A. J.; Pfefferkorn, E. R.; Boothroyd, J. C.

1992-01-01

156

Advanced microinstrumentation for rapid DNA sequencing and large DNA fragment separation  

SciTech Connect

Our efforts to develop novel technology for a rapid DNA sequencer and large fragment analysis system based upon gel electrophoresis are described. We are using microfabrication technology to build dense arrays of high speed micro electrophoresis lanes that will ultimately increase the sequencing rate of DNA by at least 100 times the rate of current sequencers. We have demonstrated high resolution DNA fragment separation needed for sequencing in polyacrylamide microgels formed in glass microchannels. We have built prototype arrays of microchannels having up to 48 channels. Significant progress has also been made in developing a sensitive fluorescence detection system based upon a confocal microscope design that will enable the diagnostics and detection of DNA fragments in ultrathin microchannel gels. Development of a rapid DNA sequencer and fragment analysis system will have a major impact on future DNA instrumentation used in clinical, molecular and forensic analysis of DNA fragments.

Balch, J.; Davidson, J.; Brewer, L.; Gingrich, J.; Koo, J.; Mariella, R.; Carrano, A.

1995-01-25

157

Clinical aspects of sperm DNA fragmentation detection and male infertility  

Microsoft Academic Search

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose

Donald P. Evenson; Regina Wixon

2006-01-01

158

Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases.  

PubMed

There are numerous restriction endonucleases (ENases) which are known never to achieve total cleavage of certain unmethylated target DNAs. In addition to EcoRII we found seven ENases (AtuBI, Cfr9I, Eco57I, Ksp632I, NaeI, NarI, and SauBMKI) that were stimulated by oligodeoxyribonucleotide (oligo) duplexes containing enzyme-specific recognition sequences to cut the target DNAs much more efficiently and in most cases even to completion. These enzymes are class-II and class-IIS Enases isolated from different bacterial species and possess a varying number of specific sites in the refractory DNA substrates. For DNA analysis and large-scale preparation of certain restriction fragments where complete digestions are essential we recommend taking into account the fact that various ENases can be activated by specific oligo duplexes to drive restriction digestions to completion. PMID:8385888

Reuter, M; Kupper, D; Pein, C D; Petrusyte, M; Siksnys, V; Frey, B; Krüger, D H

1993-03-01

159

Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch.  

PubMed

During male meiotic prophase in mammals, X and Y are in a largely unsynapsed configuration, which is thought to trigger meiotic sex chromosome inactivation (MSCI). In avian species, females are ZW, and males ZZ. Although Z and W in chicken oocytes show complete, largely heterologous synapsis, they too undergo MSCI, albeit only transiently. The W chromosome is already inactive in early meiotic prophase, and inactive chromatin marks may spread on to the Z upon synapsis. Mammalian MSCI is considered as a specialised form of the general meiotic silencing mechanism, named meiotic silencing of unsynapsed chromatin (MSUC). Herein, we studied the avian form of MSUC, by analysing the behaviour of the peculiar germline restricted chromosome (GRC) that is present as a single copy in zebra finch spermatocytes. In the female germline, this chromosome is present in two copies, which normally synapse and recombine. In contrast, during male meiosis, the single GRC is always eliminated. We found that the GRC in the male germline is silenced from early leptotene onwards, similar to the W chromosome in avian oocytes. The GRC remains largely unsynapsed throughout meiotic prophase I, although patches of SYCP1 staining indicate that part of the GRC may self-synapse. In addition, the GRC is largely devoid of meiotic double strand breaks. We observed a lack of the inner centromere protein INCENP on the GRC and elimination of the GRC following metaphase I. Subsequently, the GRC forms a micronucleus in which the DNA is fragmented. We conclude that in contrast to MSUC in mammals, meiotic silencing of this single chromosome in the avian germline occurs prior to, and independent of DNA double strand breaks and chromosome pairing, hence we have named this phenomenon meiotic silencing prior to synapsis (MSPS). PMID:20162291

Schoenmakers, Sam; Wassenaar, Evelyne; Laven, Joop S E; Grootegoed, J Anton; Baarends, Willy M

2010-02-17

160

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism  

Microsoft Academic Search

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different

S. B. Shivachandra; A. A. Kumar; R. Gautam; S. Joseph; M. K. Saxena; P. Chaudhuri; S. K. Srivastava

2006-01-01

161

Aggressive oligodendroglioma predicted by chromosome 10 restriction fragment length polymorphism analysis. Case study  

Microsoft Academic Search

Summary Oligodendrogliomas are indolent brain tumors with mean postoperative survival of about 5 years. However, the range of postoperative survivals is wide, suggesting that these tumors are heterogeneous in their biologic behavior. Using restriction fragment length polymorphism (RFLP) analysis, we studied a case of an oligodendroglioma with loss of chromosome 10 sequences, a finding that has only been reported in

Julian K. Wu; Rebecca D. Folkerth; Zhen Ye; Basil T. Darras

1993-01-01

162

Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism of gap Gene  

PubMed Central

Oligonucleotide primers specific for the Staphylococcus aureus gap gene were previously designed to identify 12 Staphylococcus spp. by PCR. In the present study, AluI digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24 Staphylococcus spp. to be identified with high specificity.

Yugueros, Javier; Temprano, Alejandro; Sanchez, Maria; Luengo, Jose Maria; Naharro, German

2001-01-01

163

Discrimination of Human Pathogenic Subspecies of Francisella tularensis by Using Restriction Fragment Length Polymorphism  

Microsoft Academic Search

Received 5 August 2002\\/Returned for modification 14 September 2002\\/Accepted 24 October 2002 We describe the use of two insertion sequence elements (ISFtu1 and ISFtu2) in Francisella tularensis to type strains by restriction fragment length polymorphism (RFLP). The RFLP profiles of 17 epidemiologically unrelated isolates were determined and compared. Our results showed that RFLP profiles can be used to assign F.

Longzhu Cui; Xiaoxue Ma; Katsuhiro Sato; Keiko Okuma; Fred C. Tenover; Elsa M. Mamizuka; Curtis G. Gemmell; Mi-Na Kim; Vivian Ferraz; Keiichi Hiramatsu; Clifford G. Clark; Tamara M. A. C. Kruk; Louis Bryden; Yolanda Hirvi; Rafiq Ahmed; Frank G. Rodgers; Christophe Cordevant; Jane S. Tang; David Cleland; Marc Lange; Rebecca Thomas; Anders Johansson; Brendan Neeson; Karen Isherwood; Anders Sjöstedt; Jill Ellis; Richard W. Titball

2003-01-01

164

THE FIRST DECADE OF TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (T-RFLP) IN MICROBIAL ECOLOGY  

Microsoft Academic Search

Terminal restriction fragment length polymorphism (T-RFLP) was introduced to environmental microbiology only a decade ago but it soon became a molecular tool of choice, due to its high throughput and phylogenetic resolution. Fierce discussions accompanied the new method leading to sophistication of the data preparation, acquisition, manipulation and standardization of analysis. Consequently, numerous approaches were proposed at various steps and

Bla STRES

2006-01-01

165

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus : a mosaic of eliminated elements  

Microsoft Academic Search

.   The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ\\u000a line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments\\u000a of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments

Yuji Goto; Souichirou Kubota; Sei-ichi Kohno

1998-01-01

166

Distribution of DNA fragment sizes after irradiation with ions  

NASA Astrophysics Data System (ADS)

Ionizing radiation is responsible for production of double-strand breaks (DSBs) in a DNA structure. In contrast to sparsely ionizing radiation, densely ionizing radiation produces DSBs that are non-randomly distributed along the DNA molecule and can form clusters of various size. The paper discusses minimalistic models that describe observable patterns of fragment length in DNA segments irradiated with heavy ions and applies the formalism to interpret the recent experimental data collected by use of atomic force microscope (AFM).

Gudowska-Nowak, E.; Psonka-Anto?czyk, K.; Weron, K.; Elsässer, T.; Taucher-Scholz, G.

2009-11-01

167

Selective Binding of anti-DNA Antibodies to Native dsDNA Fragments of Differing Sequence  

PubMed Central

Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs) has been shown. Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

Uccellini, Melissa B.; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A.

2012-01-01

168

Restriction mapping of DNA stretched in nanofluidic devices  

NASA Astrophysics Data System (ADS)

We present sequence-specific restriction mapping of single DNA molecules in nanofabricated channels. In these channels, DNA is linearized and stretched to up to 3/4 of its contour length, permitting attribution of the cutting sites to specific regions in the genetic code. In order to extract the highest amount of information from a single molecule, the restriction activity has to be localized to the nanochannel region of macroscopic devices comprising nanofluidic and microfluidic components. This regulation of digestion activity is provided by management of magnesium ions, a necessary co-factor for most restriction enzymes. The magnesium is released from a photosensitive chelator by UV photolysis. We will discuss the possibility of applying this technique to yeast DNA as a step in genome mapping.

Riehn, Robert; Wang, Yan Mei; Austin, Robert H.; Lu, Manchun; Cox, Edward C.

2004-03-01

169

Restriction site patterns in the ribosomal DNA of camelidae.  

PubMed

The restriction map of rDNA from South American camelids and the Bactrian camel was analyzed by digestion of high-molecular-weight DNA with endonucleases EcoRI,BamHI and the two combined followed by Southern blot hybridization with probes for the 18S and 28S rDNA sequences. We scored a total of 17 restriction sites, six of which were mapped conserved in all the species. The other eleven corresponded to spacer regions and revealed variations between these taxa. The study showed that the two groups differ in the length of the internal transcribed spacer. Also they showed the existence of two regions of fast evolution on the opposite termini of the external spacer. A restriction site present at low frequency in the non-transcribed spacer of guanaco and llama was the only difference encountered within the South American group. PMID:7958935

Semorile, L C; Crisci, J V; Vidal-Rioja, L

1994-01-01

170

Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis.  

PubMed Central

Campylobacter pyloridis isolates recovered from gastric biopsy specimens of 16 patients were examined by restriction endonuclease DNA analysis with HindIII. For 8 of these 16 patients two different isolates were compared to study the persistence of the colonizing strains and the stability of their DNA digest patterns during a period of 2 years (two patients), the identity or nonidentity of different colony types within one culture (two patients), and the nature of the relapses after apparently successful antibacterial therapy (four patients). The isolates from the 16 patients all produced different DNA digest patterns. Comparison of the two different isolates recovered from the same patients showed that these isolates were identical in all eight cases. Laboratory subculturing of a C. pyloridis strain (10 times) did not change its DNA digest pattern. These results indicate the stability of the DNA digest patterns and a marked variability of these patterns among isolates from different patients. Using restriction endonuclease DNA analysis, we found the persistence in the stomach of the same C. pyloridis strain during a period of 2 years and the identity of different colony types within one culture. The relapses after apparently successful antibacterial treatment could be attributed to recrudescence rather than reinfection. Restriction endonuclease DNA analysis is a sensitive and useful method for identifying C. pyloridis isolates. Images

Langenberg, W; Rauws, E A; Widjojokusumo, A; Tytgat, G N; Zanen, H C

1986-01-01

171

Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection.  

PubMed

Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments. PMID:20307487

Protozanova, Ekaterina; Zhang, Meng; White, Eric J; Mollova, Emilia T; Broeck, Dirk Ten; Fridrikh, Sergey V; Cameron, Douglas B; Gilmanshin, Rudolf

2010-03-20

172

Temporal Patterns of Nucleotide Misincorporations and DNA Fragmentation in Ancient DNA  

Microsoft Academic Search

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary

Susanna Sawyer; Johannes Krause; Katerina Guschanski; Vincent Savolainen; Svante Pääbo

2012-01-01

173

Repair of base alkylation damage in targeted restriction endonuclease sequences of plasmid DNA.  

PubMed

Sequence specific ethylation damage and repair of ethyl-adducts in selected restriction endonuclease recognition sites within p220-ras plasmid DNA was assessed by a modified Southern blotting coupled immunoprobing technique. In situ UV irradiation of DNA in gels clearly ameliorated the immunodetection of minute amounts of facultative fragments generated due to inhibition of enzyme cleavage site by covalent alkylation modification of the cognate sites. Specific and quantitative localization of induced facultative fragments was achieved in as low as 1 ng of DNA digest corresponding to a peak intensity below 0.1 absorbance unit upon laser scanning. An ENU dose dependent increase in the intensity of representative 7.1 and 7.7 kb facultative fragments was observed as a result of cleavage block at EcoRI (G/ATTC) and BamHI (G/GATCC) restriction endonuclease sites, respectively. To determine the repair in prokaryotic cells, the half-life of repairable alkyl-adducts was assessed in plasmid DNA established in various Escherichia coli strains as a function of post-treatment incubation time in the recovery medium. The repair is indicated by the gradual disappearance of the 7.1, 7.7, 11.9 and 5.5 kb facultative fragments within the wild-type and mutant E. coli strains. The ethyl-adducts within EcoRI and BamHI restriction sites were effectively lost from the target DNA in repair-proficient E. coli with an estimated t1/2 of approximately 40 min. However, decreased overall rate and at least 2.2-times lesser extent of repair was observed in the repair-deficient (ada+ogt-) and (ada-ogt+) cells. No measurable repair was noticed in alkyltransferase defective double mutant (ada-ogt-) even after 2 h of post-treatment incubation. The repair of ethyl-adducts at NotI site (GC/GGCCGC) in 5.5 kb facultative fragment occurred at a relatively faster rate (t1/2 of 27 min) in wild-type bacteria. A 1.5-fold slower repair of ethyl-adducts in BamHI and EcoRI sequences containing G/G and A/G at their cleavage sites was observed compared to C/G in NotI sequence. These results demonstrate the regioselective induction of alkyl-adducts in ethylated DNA and their differential repair in E. coli due to varied efficiency of the repair enzymes for promutagenic DNA base lesions present in different sequence context. PMID:7548206

Musarrat, J; Arezina-Wilson, J; Wani, A A

1995-09-19

174

Restriction fragment length polymorphism species-specific patterns in the identification of white truffles  

Microsoft Academic Search

A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction

Luana Bertini; Lucia Potenza; Alessandra Zambonelli; Antonella Amicucci; Vilberto Stocchi

1998-01-01

175

The biochemical and cytological characterization of Vicia faba DNA by means of MboI, AluI and Bam HI restriction endonucleases  

Microsoft Academic Search

Restriction endonucleases were employed to characterize both cytologically and electrophoretically the DNA of Vicia faba. The electrophoretic pattern of total DNA digested with AluI and MboI shows a continuous smear. Bam HI also shows a continuous smear for the bigger polynucleotide fragments and several bands in the lower part of the lane. Digestion of fixed chromosomal DNA produces metaphase longitudinal

M. Frediani; R. Mezzanotte; R. Vanni; D. Pignone; R. Cremonini

1987-01-01

176

Simple and rapid human papillomavirus genotyping method by restriction fragment length polymorphism analysis with two restriction enzymes.  

PubMed

Cervical cancer, the third most common cancer that affects women worldwide, is caused by the human papillomavirus (HPV) and is treatable when detected at an early stage. To date, more than 100 different HPV types have been described, and the development of simple, low-cost, and accurate methods to distinguish HPV genotypes is highly warranted. In this study, an HPV genotyping assay based on polymerase chain reaction (PCR) was evaluated. This method involved the use of MY09/11 primers followed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes HpyCH4V and NlaIII. Cervical specimens preserved using CytoRich Blue fluid were collected from 1,134 female volunteers for HPV detection, and 1,111 valid samples were amplified using PCR. The PCR method was sensitive enough to detect 25 copies of HPV18, and three copies of HPV16. Out of 202 PCR-positive samples, HPV genotypes were determined in 189 samples (93.6%) by this RFLP method. Results were then evaluated further by capillary sequencing method. Concordant results between the two tests were as high as 96.0%. Thirteen samples, which tested negative with RFLP, were verified as non-specific amplifications with PCR. In conclusion, this PCR-RFLP method using restriction enzymes HpyCH4V and NlaIII is simple, non-labor intensive, and is applicable for the inexpensive determination of HPV genotypes in clinical samples. PMID:23918541

Chen, Linghan; Watanabe, Ken; Haruyama, Takahiro; Kobayashi, Nobuyuki

2013-07-01

177

The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis  

Microsoft Academic Search

We report here the reconstitution of a path- way that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3. The cleaved fragments of DFF45 dissociate from the

Xuesong Liu; Peng Li; Piotr Widlak; Hua Zou; Xu Luo; WILLIAM T. GARRARD; Xiaodong Wang

1998-01-01

178

One-step isothermal assembly of DNA fragments.  

PubMed

The One-Step Isothermal DNA Assembly method allows for the efficient assembly of DNA constructs using fragments up to several hundred kilobases in as little as 15 min. Applications of this method range from the addition of promoters to expression constructs to the assembly of bacterial genome fragments. The production of circularized DNA using this method also enables the direct transformation of target organisms, bypassing intermediate transformations for plasmid propagation in those species where expression could lead to toxicity and cell death. Variations of the method allow for specific cloning tasks to be performed, as well as the use of microarray slides as a source of DNA. The level of precision and simplicity of this method makes it a valuable tool for most cloning efforts and all levels of proficiency in molecular biology. PMID:23996438

Rodrigues, Rui T L; Bayer, Travis S

2013-01-01

179

Methylation-dependent DNA restriction in Bacillus anthracis.  

PubMed

Bacillus anthracis, the causative agent of anthrax, is poorly transformed with DNA that is methylated on adenine or cytosine. Here we characterize three genetic loci encoding type IV methylation-dependent restriction enzymes that target DNA containing C5-methylcytosine (m5C). Strains in which these genes were inactivated, either singly or collectively, showed increased transformation by methylated DNA. Additionally, a triple mutant with an ~30-kb genomic deletion could be transformed by DNA obtained from Dam(+)Dcm(+)E. coli, although at a low frequency of ~10(-3) transformants/10(6)cfu. This strain of B. anthracis can potentially serve as a preferred host for shuttle vectors that express recombinant proteins, including proteins to be used in vaccines. The gene(s) responsible for the restriction of m6A-containing DNA in B. anthracis remain unidentified, and we suggest that poor transformation by such DNA could in part be a consequence of the inefficient replication of hemimethylated DNA in B. anthracis. PMID:22178763

Sitaraman, Ramakrishnan; Leppla, Stephen H

2011-12-08

180

Inheritance of restriction fragment length polymorphisms and random amplified polymorphic DNAs in coastal Douglas-fir  

Microsoft Academic Search

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29 RAPD loci was demonstrated based on single-locus segregation in a sample of F2 progeny.

K. D. Jermstad; A. M. Reem; J. R. Henifin; N. C. Wheeler; D. B. Neale

1994-01-01

181

Fiber optic system for rapid analysis of amplified DNA fragments  

NASA Astrophysics Data System (ADS)

We have developed a fiber optic sensor for rapid and direct analysis of PCR-amplified DNA fragments with minimal sample processing and real-time data readout. To accomplish this, a novel DNA-recognition system was built onto the surface of fused silica fibers. DNA fragments, labeled with a fluorophore during amplification, are bound to and detected at the fiber surface by means of evanescent wave excitation/emission. Excess unincorporated fluorescent single-stranded oligonucleotide PCR primers make only a small contribution to the signal, as the modified fiber surface only efficiently binds double-stranded DNA with the proper PCR-incorporated terminal nucleotide sequence (5'-ATGACTCAT-3'). The surface- bound double-stranded DNA recognition element utilizes a genetically engineered dimeric sequence-specific DNA binding protein. Self-assembly into the proper conformation for binding DNA occurs by means of specific interactions of the active dimer with the Fc domains of a layer of IgG molecules (antibodies) covalently attached directly to the fiber surface. The modified fiber surface is regenerated between samples by stripping away bound DNA with high salt concentrations.

Mauro, J. Matthew M.; Cao, Lynn K.; Golden, Joel P.

1996-04-01

182

Directed Transfer of Large DNA Fragments between Streptomyces Species  

Microsoft Academic Search

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically ame- nable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene

ZHIHAO HU; DAVID A. HOPWOOD; CHAITAN KHOSLA

2000-01-01

183

Short DNA fragments induce site specific recombination in mammalian cells  

Microsoft Academic Search

A defective hprt gene was corrected by homologous recombination in a lymphocyte cell line deficient in Hypoxanthine-phosphoribosyl-transferase activity (hprt). In a novel approach, only a fragment of a cDNA clone of the functional hprt gene was used to induce homologous recombination. The mutation that was corrected corresponds to a single base change in exon III of the hprt gene.

Katharina Hunger-Bertling; Petra Harrer; Wolf Bertling

1990-01-01

184

UseofLow-Frequency-Cleavage Restriction Endonucleases for DNA Analysis inEpidemiological Investigations ofNosocomial Bacterial Infections  

Microsoft Academic Search

Epidemiological investigations ofbacterial infections aregenerally based on multiple phenotypic markers that areoften difficult toverify. A more general andreliable methodisgenomic DNA analysis byrestriction endonucleases. However, thecommonlyusedendonucleases produce toomany fragments forcorrect separation byagaroseelectrophoresis. Incontrast, simple electrophoretic patterns areobtained after genomic DNA digestion bylow-frequency-cleavage restriction endonucleases andpulsed-field gelelectrophoresis, making iteasier tocompare numerous strains fromthesame species. Thistechnique was usedtoinvestigate an Acinetobacter calcoaceticus outbreak ina

ANNICK ALLARDET-SERVENT; NICOLE BOUZIGES; MARIE-JOSÉE CARLES-NURIT; GISČLE BOURG; ANNE GOUBY; MICHEL RAMUZ

1989-01-01

185

Assessment of Genetic Diversity in Oil Palm ( Elaeis guineensis Jacq.) using Restriction Fragment Length Polymorphism (RFLP)  

Microsoft Academic Search

A total of 359 accessions of oil palm (Elaeis guineensis Jacq.) originating from 11 African countries (Nigeria, Cameroon, Congo DR, Tanzania, Angola, Senegal, Sierra Leone, Guinea,\\u000a Ghana, Madagascar and Gambia) were characterized using the RFLP method using the standard Deli dura as the check. Genomic DNA from each sample was digested using five restriction enzymes and hybridized with four oil

N. Rajanaidu; A. H. Zakri; S. C. Cheah

2006-01-01

186

Comparative Typing of Campylobacter jejuni by Heat-Stable Serotyping and PCR-Based Restriction Fragment Length Polymorphism Analysis  

PubMed Central

Campylobacter jejuni has become the most common bacterial cause of human gastroenteritis worldwide. Rapid, discriminatory typing methods are required to identify potential clusters of infections. The major disadvantage of the well-evaluated and widely used Penner heat-stable serotyping method is the high level of nontypeability. The correlation of the types determined by the Penner heat-stable serotyping method and PCR-based restriction fragment length polymorphism (RFLP) analysis of the lipooligosaccharide (LOS) biosynthesis genes of C. jejuni was studied with 149 C. jejuni strains. Of these strains, 79 were patient strains belonging to 25 Penner serotypes, 60 were nontypeable patient strains, and 10 were reference strains. A 9.6-kb DNA fragment of the LOS gene cluster was amplified and digested with the restriction enzymes HhaI and DdeI. Altogether, 39 different RFLP types (including 30 HhaI profiles and 32 DdeI profiles) were identified. Type Hh1Dd1 was the most common type, with 36% of the strains and strains of 12 serotypes being of this type. A high level of discrimination was obtained, and a correlation between the Penner serotypes and the PCR-RFLP types could be seen. Also, variation in the LOS biosynthesis genes within a single Penner serotype was found. Although the PCR-RFLP method may not be sufficient to compensate for Penner serotyping, it can give valuable information about nontypeable strains and further characterize strains of common serotypes.

Nakari, Ulla-Maija; Laaksonen, Katja; Korkeila, Maija; Siitonen, Anja

2005-01-01

187

Automated DNA fragments recognition and sizing through AFM image processing.  

PubMed

This paper presents an automated algorithm to determine DNA fragment size from atomic force microscope images and to extract the molecular profiles. The sizing of DNA fragments is a widely used procedure for investigating the physical properties of individual or protein-bound DNA molecules. Several atomic force microscope (AFM) real and computer-generated images were tested for different pixel and fragment sizes and for different background noises. The automated approach minimizes processing time with respect to manual and semi-automated DNA sizing. Moreover, the DNA molecule profile recognition can be used to perform further structural analysis. For computer-generated images, the root mean square error incurred by the automated algorithm in the length estimation is 0.6% for a 7.8 nm image pixel size and 0.34% for a 3.9 nm image pixel size. For AFM real images we obtain a distribution of lengths with a standard deviation of 2.3% of mean and a measured average length very close to the real one, with an error around 0.33%. PMID:16379368

Ficarra, Elisa; Benini, Luca; Macii, Enrico; Zuccheri, Giampaolo

2005-12-01

188

The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation  

Microsoft Academic Search

Sperm DNA fragmentation is being increasingly rec- ognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm

JOSE LUIS FERNANDEZ; LOURDES MURIEL; MARIA TERESA RIVERO; VICENTE GOYANES; ROSANA VAZQUEZ; JUAN G. ALVAREZ

2003-01-01

189

Detection of Oxazolidinone-Resistant Enterococcus faecalis and Enterococcus faecium Strains by Real-Time PCR and PCR-Restriction Fragment Length Polymorphism Analysis  

Microsoft Academic Search

A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.

Neil Woodford; Luke Tysall; Cressida Auckland; Mark W. Stockdale; Andrew J. Lawson; Rachel A. Walker; David M. Livermore

2002-01-01

190

Differentiation of soybean-nodulating Bradyrhizobium USDA strains using restriction fragment length polymorphism analysis of 23S–5S rRNA genes  

Microsoft Academic Search

Cluster analysis of the electrophoresis patterns from polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of approximately 3 kbp of the 23S–5S rRNA genes (rDNA) of 21 Bradyrhizobium USDA strains and a Sinorhizobium fredii type strain was conducted to determine the usefulness of these genes for differentiating soybean-nodulating bacteria. The PCR-RFLP analysis of 23S–5S rDNAs separately digested by each of the four

Yuichi Saeki; Tadashi Murata; Takeo Yamakawa; Shoichiro Akao

2007-01-01

191

Evaluation of PCR-Restriction Profile Analysis and IS2404 Restriction Fragment Length Polymorphism and Amplified Fragment Length Polymorphism Fingerprinting for Identification and Typing of Mycobacterium ulcerans and M. marinum  

PubMed Central

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marinum (n = 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3? end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270–273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.

Chemlal, K.; Huys, G.; Fonteyne, P.-A.; Vincent, V.; Lopez, A. G.; Rigouts, L.; Swings, J.; Meyers, W. M.; Portaels, F.

2001-01-01

192

Distinguishing Heterodera filipjevi and H. avenae using polymerase chain reaction-restriction fragment length polymorphism and cyst morphology.  

PubMed

The cereal cyst nematodes Heterodera filipjevi and H. avenae impede wheat production in the Pacific Northwest (PNW). Accurate identification of cyst nematode species and awareness of high population density in affected fields are essential for designing effective control measures. Morphological methods for differentiating these species are laborious. These species were differentiated using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS)-ribosomal (r)DNA with up to six restriction endonucleases (TaqI, HinfI, PstI, HaeIII, RsaI, and AluI). The method was validated by inspecting underbridge structures of cyst vulval cones. Grid soil sampling of an Oregon field infested by both species revealed that H. filipjevi was present at most of the infested grid sites but mixtures of H. avenae and H. filipjevi also occurred. These procedures also detected and differentiated H. filipjevi and H. avenae in soil samples from nearby fields in Oregon and H. avenae in samples from Idaho and Washington. Intraspecific polymorphism was not observed within H. filipjevi or PNW H. avenae populations based on the ITS-rDNA. However, intraspecific variation was observed between H. avenae populations occurring in the PNW and France. Methods described here will improve detection and identification efficiencies for cereal cyst nematodes in wheat fields. PMID:20128694

Yan, Guiping; Smiley, Richard W

2010-03-01

193

Restriction enzyme analysis of mitochondrial DNA of the Aspergillus flavus group: A. flavus, A. parasiticus, and A. nomius.  

PubMed Central

Mitochondrial DNA restriction fragment length polymorphisms were identified that clearly distinguish Aspergillus flavus, A. parasiticus, and A. nomius. Mitochondrial DNAs of A. flavus and A. parasiticus were found to be circular, and their size was estimated size to be 32 kilobases. A restriction map was constructed for the mitochondrial genome of an A. parasiticus isolate by using four restriction endonucleases. Four genes tested were found to have the same order as in the mitochondrial genome of A. nidulans. The mitochondrial genome of A. nomius was estimated to be 33 kilobases. Images

Moody, S F; Tyler, B M

1990-01-01

194

Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQwl and DQw3 alleles of the HLA-D region  

SciTech Connect

Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ..beta.., DQ..cap alpha.., and DR..beta.. cDNA probes. Hybridization with the DQ..beta.. probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ..cap alpha.. and DR..beta.. probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.

Szafer, F.; Brautbar, C.; Tzfoni, E.; Frankel, G.; Sherman, L.; Cohen, I.; Hacham-Zadeh, S.; Aberer, W.; Tappeiner, G.; Holubar, K.; Steinman, L.

1987-09-01

195

A novel antitumor compound, NC190, induces topoisomerase II-dependent DNA cleavage and DNA fragmentation  

Microsoft Academic Search

A novel benzophenazine derivative, NC-190, is a potent antitumor compound. NC-190 has been shown to inhibit the DNA strand-passing\\u000a activity of DNA topoisomerase II. We investigated further the mode of action of NC-190 against DNA topoisomerase II and DNA\\u000a fragmentation. NC-190 inhibited the decatenation activity of purified topoisomerase II, but had only a weak inhibitory effect\\u000a against topoisomerase I. A

Takehiro Yamagishi; Shiro Nakaike; Tomotake Ikeda; Hisao Ikeya; Susumu Otomo

1996-01-01

196

Species identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism analysis.  

PubMed

Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were used to detect and differentiate four pathogenic species (Mycoplasma gallisepticum, M. iowae, M. meleagridis, and M. synoviae) and ten nonpathogenic species of avian mycoplasma. A sequence of 1026 base pairs within the gene for 16S ribosomal RNA (16S rRNA) from avian mycoplasmas was successfully amplified by PCR with oligonucleotide primers (M16SPCR5' and M16SPCR3') common to all avian mycoplasmas tested. Restriction endonucleases (REs) with unique restriction sites, selected by computer-assisted analysis of known sequences of the amplified segment of avian mycoplasma, were then used to digest the PCR products. After electrophoresis of the resulting RE fragments, the RFLP patterns were compared. Combinations of up to six REs (HpaI, HhaI, HaeIII, HphI, FokI, and NlaIV) produced unique RFLP patterns by which the 14 species of avian mycoplasmas could be differentiated. The newly classified avian species M. imitans was also investigated by this method; M. imitans and M. gallisepticum gave identical RFLP patterns with the REs used in this study. The results obtained by the PCR and RFLP analysis were in agreement with current methods for species identification of avian mycoplasmas. PMID:7677664

Fan, H H; Kleven, S H; Jackwood, M W; Johansson, K E; Pettersson, B; Levisohn, S

197

Restriction fragment length polymorphisms in the VP2 gene of infectious bursal disease viruses.  

PubMed

Infectious bursal disease viruses (IBDVs) were examined for restriction fragment length polymorphisms in a fraction of the VP2 gene with the use of the reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay. The restriction enzymes BstNI and Mbol were used to obtain RFLP results. A third enzyme, StyI, was tested, but its utility for differentiation of IBDV strains was limited. Thirteen vaccine viruses and five IBDV strains that were previously characterized were placed into five molecular groups. Two groups contained viruses described as being classic strains, and two groups contained viruses described as being variant strains. The fifth group contained both classic and variant strains. The RFLP observed for the serotype 2 IBDV strain OH was unlike any of the RFLPs observed in viruses in the five molecular groups. Seven IBDV strains from commercially reared chickens in the United States, Mexico, Puerto Rico, and Thailand were tested in the RT/PCR-RFLP assay to determine if they were similar to the commercial IBDV vaccine strains tested. These viruses were selected because they were associated with lesions in the bursa of chickens that should have been protected by maternal antibodies or active immunity. Each of the viruses tested contained a unique RFLP compared with the IBDV strains and vaccine viruses examined in this study and, thus, did not fit into any of the five molecular groups. These viruses also were distinguishable from each other. PMID:9356709

Jackwood, D J; Sommer, S E

198

Qualitative and quantitative analysis of DNA fragmentation using digital imaging.  

PubMed

Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-alpha, transforming growth factor-beta1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software. PMID:9245431

Vodovotz, Y; Hsing, A; Cook, J A; Miller, R W; Wink, D A; Ritt, D M; Mitchell, J B; Danielpour, D

1997-08-01

199

[Analysis of microbial diversity of nitrifying bacteria by terminal restriction fragment length polymorphism].  

PubMed

We analyzed the microbial diversity and quantity of nitrifying bacteria in the enrichment reactor by Terminal Restriction Fragment Length Polymorphism (T_RFLP), a cultured-independent molecular technique. The result indicated that nitrobacteria enriched the best, and the diversity index decreased 62.80% compared with the initial data. Nitrobacteria were predominant in the reactor. Meanwhile, we studied the microbial diversity before and after adding Nitrobacteria into shrimp ponds, and analyzed several major bacterial species that existed stably in the pond. According to the analysis by T_RFLP program, species including Brevibacillus brevis, Microbacterium lactium, Azoarcus indigens and Bordetella holmesii were the dominant bacteria in the ponds. PMID:20575436

Lin, Weitie; Zhu, Yanan

2010-04-01

200

Misidentification of a genomovar of Burkholderia cepacia by recA restriction fragment length polymorphism  

PubMed Central

An 8 year old girl with cystic fibrosis presented with a pulmonary exacerbation from which Burkholderia cepacia was cultured. Subsequent polymerase chain reaction restriction fragment length polymorphism analysis of the recA gene suggested the presence of B cepacia Genomovar V (Burkholderia vietnamiensis); however, on subsequent sequence typing, this isolate was confirmed as B cepacia Genomovar IIIb. This report outlines the potential difficulties in the correct characterisation of the various genomovars within the B cepacia complex of organisms, which has particularly important implications for patient segregation and infection control.

Moore, J E; Millar, B C; Xu, J; Crowe, M; Redmond, A O B; Elborn, J S

2002-01-01

201

Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay.  

PubMed

The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP, based identification procedures of 17 different field isolates agreed with those obtained by conventional methods. PMID:9451458

Kiss, I; Matiz, K; Kaszanyitzky, E; Chávez, Y; Johansson, K E

1997-10-31

202

Elimination of Bacterial DNA from Taq DNA Polymerases by Restriction Endonuclease Digestion  

Microsoft Academic Search

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S

NORA M. CARROLL; PETER ADAMSON; NARCISS OKHRAVI

1999-01-01

203

DNA fragmentation in leukocytes following subacute lowdose nerve agent exposure  

Microsoft Academic Search

The objective of the present study was to determine levels of DNA fragmentation in blood leukocytes from guinea pigs by single-cell gel electrophoresis (comet assay) after exposure to the chemical warfare nerve agent (CWNA), soman, at doses ranging from 0.1 LD 50 to 0.4 LD 50, once per day for either 5 or 10 days. Post-exposure recovery periods ranged from

J. R. Moffett; R. A. Price; S. M. Anderson; M. L. Sipos; A. V. Moran; F. C. Tortella; J. R. Dave

2003-01-01

204

Regulation of DNA fragmentation: the role of caspases and phosphorylation.  

PubMed

DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation. PMID:21182594

Kitazumi, Ikuko; Tsukahara, Masayoshi

2010-12-23

205

Characterization by restriction endonuclease analysis and DNA hybridization using IS 900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities  

Microsoft Academic Search

DNA of 90 mycobactin-dependent strains of Mycobacterium paratuberculosis, isolated in 9 countries, was digested with restriction endonuclease PstI and hybridized with a DNA fragment containing insertion sequence IS900. Bovine strains (n = 73) were isolated from 61 animals in 17 herds, ovine strains (n = 15) from 13 animals in 3 herds and the set was completed by 1 caprine

I. Pavlík; L. Bej?ková; M. Pavlas; Z. Rozsypalová; S. Kosková

1995-01-01

206

Identification of Major Streptococcal Species by rrn-Amplified Ribosomal DNA Restriction Analysis  

PubMed Central

Amplified ribosomal DNA restriction analysis (rrn-ARDRA) is based on PCR amplification and restriction of a fragment of rRNA genes including 16S and 23S genes and the intergenic spacer. rrn-ARDRA was evaluated for the identification of species within the genus Streptococcus. A total of 148 type and reference strains of pyogenic, oral, and group D streptococci were examined in order to construct a database for identification of streptococci. The amplified product was a single band approximately 4,500 bp long. This amplicon was digested separately with three (HhaI, MboII, and Sau3A) restriction endonucleases. Respectively, 27, 26, and 28 major patterns were observed after HhaI, MboII, and Sau3A restrictions. Streptococcal strains belonging to different species had different patterns or different combination of patterns. An identification system based upon a combination of the three restriction patterns in a single database was then proposed. rrn-ARDRA was successfully applied to 11 clinical isolates whose identification to the species level was difficult to obtain by phenotypic analysis. Using a database of well-characterized strains, rrn-ARDRA is a powerful method for the identification of streptococcal isolates.

Schlegel, Laurent; Grimont, Francine; Grimont, Patrick A. D.; Bouvet, Anne

2003-01-01

207

DNA Flexibility Studied by Covalent Closure of Short Fragments into Circles  

NASA Astrophysics Data System (ADS)

The ring closure probability, or j factor, has been measured for DNA restriction fragments of defined sequence bearing EcoRI cohesive ends and ranging in size from 126 to 4361 base pairs (bp). The j factor is defined as the ratio of the equilibrium constants for cyclization and for bimolecular association via the cohesive ends. The end-joining reactions are fast compared to covalent closure of the cohesive ends by T4 DNA ligase. The rate of ligase closure is shown to be proportional to the equilibrium fraction of DNA molecules with joined cohesive ends, both in cyclization and in bimolecular association reactions. The j factor changes by less than 10-fold between 242 and 4361 bp, whereas it decreases by more than 100-fold between 242 and 126 bp as the DNA reaches the size range of the persistence length (150 bp). As regards ring closure, short DNA fragments are surprisingly flexible. These data are in good agreement with predictions by others for the ring closure probability of a wormlike chain.

Shore, David; Langowski, Jorg; Baldwin, Robert L.

1981-08-01

208

Intrauterine calorie restriction affects placental DNA methylation and gene expression.  

PubMed

Maternal nutrient restriction causes the development of adult onset chronic diseases in the intrauterine growth restricted (IUGR) fetus. Investigations in mice have shown that either protein or calorie restriction during pregnancy leads to glucose intolerance, increased fat mass, and hypercholesterolemia in adult male offspring. Some of these phenotypes are shown to persist in successive generations. The molecular mechanisms underlying IUGR remain unclear. The placenta is a critical organ for mediating changes in the environment and the development of embryos. To shed light on molecular mechanisms that might affect placental responses to differing environments we examined placentas from mice that had been exposed to different diets. We measured gene expression and whole genome DNA methylation in both male and female placentas of mice exposed to either caloric restriction or ad libitum diets. We observed several differentially expressed pathways associated with IUGR phenotypes and, most importantly, a significant decrease in the overall methylation between these groups as well as sex-specific effects that are more pronounced in males. In addition, a set of significantly differentially methylated genes that are enriched for known imprinted genes were identified, suggesting that imprinted loci may be particularly susceptible to diet effects. Lastly, we identified several differentially methylated microRNAs that target genes associated with immunological, metabolic, gastrointestinal, cardiovascular, and neurological chronic diseases, as well as genes responsible for transplacental nutrient transfer and fetal development. PMID:23695884

Chen, Pao-Yang; Ganguly, Amit; Rubbi, Liudmilla; Orozco, Luz D; Morselli, Marco; Ashraf, Davin; Jaroszewicz, Artur; Feng, Suhua; Jacobsen, Steve E; Nakano, Atsushi; Devaskar, Sherin U; Pellegrini, Matteo

2013-05-21

209

Detection of Irradiated Food: DNA Fragmentation in Grapefruits  

NASA Astrophysics Data System (ADS)

Employing the simple microgel electrophoresis of single cells - `comet assay' - on grapefruit seeds enabled a rapid identification of irradiated fruits. Fruits were exposed to radiation doses of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 kGy covering the range of potential commercial irradiation for insect disinfestation and quarantine purposes. Seeds were isolated, crushed, and the cells embedded in an agarose layer. After lysis of the cells, they were subjected to microgel electrophoresis for 2.5 minutes, and then stained. Fruits irradiated with 0.2 kGy and higher doses showed typical DNA fragmentation, the DNA fragments stretching or migrating out of the cells forming a tail towards the anode, giving the damaged cells an appearance of a comet. With increasing dose a longer extension of the DNA from the nucleus towards the anode is observed. Undamaged cells will appear as intact nuclei without tails. The DNA comet assay is thus a rapid and inexpensive screening technique to detect irradiated grapefruits. Suspected samples may subsequently be analysed by officially validated methods for detection of irradiated foods.

Delincée, Henry

1998-06-01

210

Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis.  

PubMed Central

We developed and studied a molecular typing approach for Campylobacter spp. with restriction fragment length polymorphism (RFLP) analysis of the flagellin gene flaA in C. jejuni. Using polymerase chain reaction, we amplified the flaA gene from strains comprising different HL:O serotypes by using a primer set directed at the conserved 5' and 3' flaA gene sequence to generate a 1.7-kb amplicon. The amplicon was further digested with the restriction enzyme DdeI, and the fragments generated were analyzed by agarose gel electrophoresis. In 43 non-outbreak strains of six common HL serotypes (HL 1, 2, 4, 5, 9, and 36) in the United States, 18 RFLP patterns were observed. In U.S. outbreak strains previously studied by 10 other typing methods, flaA typing correlated with the HL serotype within each outbreak, and six additional flaA types were identified. Our results suggest that RFLP analysis of the flaA gene from Campylobacter spp. has sufficient discrimination to be useful as a practical typing method for clinical and epidemiologic investigations. Images

Nachamkin, I; Bohachick, K; Patton, C M

1993-01-01

211

Resolution of recent radiations within three evolutionary lineages of felidae using mitochondrial restriction fragment length polymorphism variation  

Microsoft Academic Search

Patterns of mitochondrial restriction fragment length polymorphism (RFLP) variation were used to resolve more recent relationships among the species of the Felidae ocelot lineage, domestic cat lineage, and pantherine lineage. Twenty-five of 28 restriction enzymes revealed site variation in at least 1 of 21 cat species. The ocelot lineage was resolved into three separate sistertaxa groups: Geoffroy's cat (Oncifelis geoffroyi)

Warren E. Johnson; Peter A. Dratch; Janice S. Martenson; Stephen J. O'Brien

1996-01-01

212

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm2a region of chromosome 9 in tomato  

Microsoft Academic Search

A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number

Martin W. Ganal; Nevin D. Young; Steven D. Tanksley

1989-01-01

213

Identification of 54 Mycobacterial Species by PCR-Restriction Fragment Length Polymorphism Analysis of the hsp65 Gene  

Microsoft Academic Search

A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction

Francesca Brunello; Marco Ligozzi; Emanuela Cristelli; Stefano Bonora; Enrico Tortoli; Roberta Fontana

2001-01-01

214

Detection of Mycoplasma gallisepticum, M. synoviae, and M. iowae by multi-species polymerase chain reaction and restriction fragment length polymorphism.  

PubMed

A single set of oligonucleotide primers was designed from known 16S ribosomal RNA (rRNA) sequences of Mycoplasma gallisepticum (MG), M. synoviae (MS), and M. iowae (MI). This set of primers selectively amplifies a 780-base-pair DNA fragment within the 16S rRNA gene of MG, MS, and MI but does not amplify other avian mycoplasmas or other bacteria. The detection limit of the multi-species polymerase chain reaction (PCR) was approximately 100 mycoplasma (MG, MS, MI) colony-forming units per PCR reaction. The PCR product was differentiated by restriction fragment length polymorphism with the restriction enzymes HpaI, HpaII, and MboI. Preliminary results from field samples suggest that this technique could be a useful and rapid diagnostic test for the detection of these three pathogenic poultry mycoplasmas. PMID:8561747

Garcia, M; Jackwood, M W; Levisohn, S; Kleven, S H

215

Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism  

Microsoft Academic Search

Terminal restriction fragment length polymorphism (T-RFLP) can be used to assess how land use management changes the dominant\\u000a members of bacterial communities. We compared T-RFLP profiles obtained via amplification with forward primers (27, 63F) each\\u000a coupled with the fluorescently labeled reverse primer (1392R) and multiple restriction enzymes to determine the best combination\\u000a for interrogating soil bacterial populations in an agricultural

Ann-Marie Fortuna; Terence L. Marsh; C. Wayne Honeycutt; William A. Halteman

216

From organic superconductors to DNA: Fragment orbital-based model  

NASA Astrophysics Data System (ADS)

A semi-empirical valence bond/Hartree-Fock (VB/HF) method is developed to calculate one- and two-electron interactions between molecular fragments in conducting supramolecular stacks. This fragment orbital-based formalism allows for consistent extraction of an effective Hamiltonian defined as a "frontier orbital" model. This Hamiltonian quantitatively describes transfer and electrostatic interactions between conducting electrons, while reducing the active space so dramatically that the electronic eigenstates of very large systems may be investigated. The capabilities of the VB/HF method are illustrated on two different supramolecular stacks involving a ?-? interacting fragment. In the first part of this study, the framework of the VB/HF method is used to evaluate the relative magnitude of the electronic interactions between conduction electrons in organic conductors and superconductors derived from Bechgaard salts. In the second part of this study, the VB/HF formalism is extended to derive an effective model for conduction holes along doped DNA double strands. Transferable intra- and intersite parameters were first evaluated from VB/HF calculations carried out on nucleoside pairs. From this interaction databank, the effective Hamiltonian of any type of nucleoside sequence can be defined. The thermalized charge distribution for a single hole delocalized along a DNA sequence containing 240 Watson-Crick pairs is then calculated and compared with the experimental yields of damage revealed by photocleavage experiments.

Castet, Frédéric; Ducasse, Laurent; Fritsch, Alain

217

Diversity analysis of magnetotactic bacteria in Lake Miyun, northern China, by restriction fragment length polymorphism.  

PubMed

Magnetotactic bacteria (MTB) synthesize intracellular nano-scale crystals of magnetite or greigite within magnetosomes. MTB are ubiquitous in limnic and marine environments. In order to understand the diversity of MTB better, sediment samples were examined from Lake Miyun near Beijing by restriction fragment length polymorphism (RFLP). First, in silico analysis was used to evaluate the effectiveness of 12 sets of restriction endonucleases for distinguishing MTB sequences retrieved from the GenBank database. It was found that the tested restriction endonucleases had different power in the ability to differentiate the operational taxonomic units (OTUs) of MTB. Specifically, of the 12 sets of enzymes, MspI plus RsaI was found to be the most effective for correctly differentiating the OTUs of selected MTB sequences and it could detect 16 OTUs with appropriate OTUmin and OTUmax values (96.7% and 97.7%, respectively). The MspI plus RsaI RFLP analysis was then utilized to investigate the diversity of MTB in Lake Miyun sediment and it identified 8 OTUs (74.5% of the whole library) as MTB. Among these, 5 were affiliated to Alphaproteobacteria, while the rest belonged to the Nitrospira phylum. Interestingly, OTUs C, D and I displayed 91.8-98.4% similarity to "Magnetobacterium bavaricum". Together, these results demonstrated that the MspI plus RsaI RFLP analysis was useful for studying the diversity and change in community composition of uncultivated MTB from environmental samples. PMID:19168303

Lin, Wei; Li, Jinhua; Schüler, Dirk; Jogler, Christian; Pan, Yongxin

2009-01-24

218

Evaluation of terminal-restriction fragment length polymorphism analysis in contrasting marine environments.  

PubMed

Terminal-restriction fragment length polymorphism (T-RFLP) analysis is widely used in microbial ecology studies. In the present study, T-RFLP analysis of PCR products digested by five restriction enzymes (AluI, HaeIII, MspI, Sau3AI and TaqI) was applied for 20 samples from three contrasting coastal environments to assess the biases associated with the choice of enzyme digestion and T-RF analysis. The five enzyme digestions produced highly variable species richness (in terms of number of T-RFs). Analysis of peak areas with a threshold of 0.5% of the total peak area, which recovered 92-96% of the total peak area, revealed different diversity indexes from the five enzyme digestions. Multidimensional scaling, based on matrices that were generated by scoring peak presence/absence and area, revealed similar bacterial community structure patterns among the 20 samples, regardless of the choice of restriction enzymes. Our results strongly argue that the choice of different digestion enzymes in the T-RFLP technique generated valid and consistent bacterial community structures but highly variable species richness and diversity indices. The biases associated with the choice of digestion enzymes needs to be evaluated carefully or at least to be addressed when using T-RFLP analysis. PMID:18503550

Zhang, Rui; Thiyagarajan, Vengatesen; Qian, Pei-Yuan

2008-05-22

219

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosome 12q.  

PubMed

Human gene mapping would be greatly facilitated if marker loci with sufficient polymorphism information content were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have used a rapid method for screening random cosmids to identify those homologous to genomic regions especially rich in restriction fragment length polymorphisms (Litt and White 1985). This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to anneal with Southern transfers by prehybridization of the probes with a vast excess of non-radioactive genomic DNA. From one cosmid (C1-11) identified by this procedure, we have isolated four single-copy probes, each of which identifies a polymorphic locus. Despite the existence of some linkage disequilibrium in this system, the polymorphism information content was computed as 0.73. Using a somatic cell hybrid mapping panel, we have mapped probes from cosmid 1-11 to human chromosome 12q. Additionally, in situ hybridization of the whole cosmid to metaphase spreads allowed more precise assignment of the locus to the region 12cen----q13. The locus revealed by probes from cosmid 1-11 has been designated D12S6. PMID:3002956

Buroker, N E; Magenis, R E; Weliky, K; Bruns, G; Litt, M

1986-01-01

220

Chloroplast DNA variability in the genus Helianthus: restriction analysis and S1 nuclease mapping of DNA-DNA heteroduplexes.  

PubMed

Chloroplast DNA (cpDNA) from 36 wild species of the genus Helianthus has been analysed with three restriction endonucleases (Bam HI, Hind III and Sst I). Out of the 71 restriction sites described on the reference cpDNA (sunflower cpDNA), three insertions/deletions and seven site modifications were detected during the survey of the other cpDNAs. Since restriction mapping showed only a very limited fraction of the DNA variability, we chose to adapt the S1 nuclease mapping technique to detect fine variations between chloroplast genomes. For this purpose, DNA-DNA heteroduplexes obtained between sunflower and wild-species DNAs were digested by S1 nuclease and the resulting mismatches were detected by classical endonuclease restriction and hybridization methods. The S1 nuclease mapping results were confirmed by sequencing one S1 nuclease-sensitive region detected between cultivated sunflower and two perennial wild-type species. As a result of these analyses, it appeared that the combination of restriction mapping and S1 nuclease mapping might be helpful to differentiate taxonomically close cytoplasms. PMID:1983299

Serror, P; Heyraud, F; Heizmann, P

1990-08-01

221

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements  

SciTech Connect

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.

Tully, D.B.; Cidlowski, J.A. (Univ. of North Carolina, Chapel Hill (USA))

1989-03-07

222

Restriction Fragment Length Polymorphism Separates Species of the Xiphinema americanum Group.  

PubMed

The Xiphinema americanum group of species is responsible for vectoring several important virus diseases to perennial crops. Variability of transmission of viruses by different species, and difficulties in separating species by morphometric measurements alone, make it essential to reassess the taxonomic position of several species in the group. The measurement of DNA sequence variability is a sensitive assay that can re-evaluate the separation of species and populations from each other. This study describes how an RFLP approach, in which the restriction sites in transcribed spacers of ribosomal repeats were detected, confirmed the separation of 16 populations of these species into X. americanum, X. rivesi, X. pacificum, and X. bricolensis. PMID:19279780

Vrain, T C

1993-09-01

223

Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains  

PubMed Central

Helicobacter pylori, bacteria that colonize the human gastric mucosa, possess a large number of genes for restriction-modification (R-M) systems, and essentially, every strain possesses a unique complement of functional and partial R-M systems. Nearly half of the H.pylori strains studied possess an active type IIs R-M system, HpyII, with the recognition sequence GAAGA. Recombination between direct repeats that flank the R-M cassette allows for its deletion whereas strains lacking hpyIIRM can acquire this cassette through natural transformation. We asked whether strains lacking HpyII R-M activity can acquire an active hpyIIRM cassette [containing a 1.4 kb kanamycin resistance (aphA) marker], whether such acquisition is DNase sensitive or resistant and whether restriction barriers limit acquisition of chromosomal DNA. Our results indicate that natural transformation and conjugation-like mechanisms may contribute to the transfer of large (4.8 kb) insertions of chromosomal DNA between H.pylori strains, that inactive or partial R-M systems can be reactivated upon recombination with a functional allele, consistent with their being contingency genes, and that H.pylori R-M diversity limits acquisition of chromosomal DNA fragments of ?1 kb.

Aras, Rahul A.; Small, Aaron J.; Ando, Takafumi; Blaser, Martin J.

2002-01-01

224

Sequence-specific modification of genomic DNA by small DNA fragments  

PubMed Central

Small DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, ?-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.

Gruenert, Dieter C.; Bruscia, Emanuela; Novelli, Giuseppe; Colosimo, Alessia; Dallapiccola, Bruno; Sangiuolo, Federica; Goncz, Kaarin K.

2003-01-01

225

Genotyping of Mycobacterium tuberculosis clinical isolates using IS6110-based restriction fragment length polymorphism analysis.  

PubMed

A number of phylogenetic studies of Mycobacterium tuberculosis have suggested a highly clonal population structure. Despite the extreme homogeneity of M. tuberculosis strains, the genome is punctuated by a number of polymorphic regions that give rise to sufficient diversity, thus forming the basis for molecular epidemiologic studies of tuberculosis. As such, insertion sequence (IS) 6110, which is unique to members of the M. tuberculosis complex and is present in variable numbers and in discrete genomic locales among strains, has been extensively used in molecular epidemiologic studies. Genotyping, using IS6110-based restriction fragment length polymorphism (RFLP), was standardized by the international community, and this has facilitated inter- and intralaboratory comparison, thereby serving as a model system for subspeciation of M. tuberculosis. When IS6110-based RFLP was used in conjunction with conventional epidemiologic data, its utility was realized. In this chapter, we discuss the basic methodology for conducting IS6110-based RFLP and analyzing the resulting hybridization profiles. PMID:19521875

Bifani, Pablo; Kurepina, Natalia; Mathema, Barun; Wang, Xiao-Ming; Kreiswirth, Barry

2009-01-01

226

Phylogenomics of caspase-activated DNA fragmentation factor  

SciTech Connect

The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans.

Eckhart, Leopold [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria)]. E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria); Tschachler, Erwin [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria)

2007-04-27

227

Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.  

PubMed

We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids. PMID:1652361

Gareis, M; Harrer, P; Bertling, W M

1991-01-01

228

PCR-based restriction fragment length polymorphism typing of Helicobacter pylori.  

PubMed Central

We applied a molecular typing approach for Helicobacter pylori that uses restriction fragment length polymorphism (RFLP) analyses of an 820-bp PCR-amplified portion of the ureC gene in H. pylori. The PCR products were digested with restriction enzyme HhaI, MboI, or MseI, and the fragments generated were analyzed by agarose electrophoresis. Among 25 independent clinical isolates, each showed a different pattern when a combination of the three RFLP patterns was used. Using this method, we studied isolates from the antrum or the body of the stomach of 14 patients before and after antibiotic therapy. Before treatment, successful isolation of H. pylori from the two sites of the stomach was possible for 12 of the 14 patients. For 10 of these 12 patients, each pair of isolates had identical RFLP profiles. For the other two patients (16.7%), however, isolates from the antrum and the body of the stomach had different RFLP profiles. Treatment was successful for 6 of the 14 patients; of the 8 patients with treatment failures, 5 had identical isolate pairs. In each case, the isolates found posttreatment were the same as the pretreatment isolates. For one of the patients who was colonized with two different isolates pretreatment, one of the isolates was identified at both sites after unsuccessful treatment. We also studied six long-term follow-up patients who had sequential biopsies at intervals of up to 5 months. Each follow-up isolate from each patient had the same RFLP profile as the initial isolate. This typing method provides a reliable and reproducible typing scheme for the study of H. pylori infections and indicates that infection with more than one H. pylori isolate is not rare. Images

Fujimoto, S; Marshall, B; Blaser, M J

1994-01-01

229

A restriction endonuclease cleavage map of mouse mitochondrial DNA.  

PubMed Central

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm. Images

Moore, K H; Johnson, P H; Chandler, S E; Grossman, L I

1977-01-01

230

Cleavage of a model DNA replication fork by a Type I restriction endonuclease  

Microsoft Academic Search

Cleavage of a DNA replication fork leads to fork restoration by recombination repair. In prokaryote cells carrying restriction-modification systems, fork passage reduces genome methylation by the modification enzyme and exposes the chromosome to attack by the restriction enzyme. Various obser- vations have suggested a relationship between the fork and Type I restriction enzymes, which cleave DNA at a distance from

Ken Ishikawa; N. Handa; Ichizo Kobayashi

2009-01-01

231

A STUDY OF PARAMETERS THAT INFLUENCE THE HPLC AND CE SEPARATION OF DOUBLE STRANDED DNA FRAGMENTS AND DNA MUTANTS  

Microsoft Academic Search

The present study evaluates HPLC and CE experimental parameters, such as column packing properties, mobile phase pH, column temperature, and gel type on the separation of DNA fragments and heteroduplexes. The results of this study show that both HPLC and CE are useful techniques for the separation of DNA fragments and for the detection of DNA mutants.Not all HPLC columns

Haleem J. Issaq; Hongyu Xu; King C. Chan

2001-01-01

232

New Threshold and Confidence Estimates for Terminal Restriction Fragment Length Polymorphism Analysis of Complex Bacterial Communities†  

PubMed Central

Terminal restriction fragment length polymorphism (T-RFLP) analysis has the potential to be useful for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables. To do this successfully, systematic variations have to be detected above method-associated noise, by standardizing data sets and assigning confidence estimates to relationships detected. We investigated the use of different standardizing methods in T-RFLP analysis of PCR-amplified 16S rRNA genes to elucidate the similarities between the bacterial communities in 17 soil and sediment samples. We developed a robust method for standardizing data sets that appeared to allow detection of similarities between complex bacterial communities. We term this the variable percentage threshold method. We found that making conclusions about the similarities of complex bacterial communities from T-RFLP profiles generated by a single restriction enzyme (RE) may lead to erroneous conclusions. Instead, the use of multiple REs, each individually, to generate multiple data sets allowed us to determine a confidence estimate for groupings of apparently similar communities and at the same time minimized the effects of RE selection. In conjunction with the variable percentage threshold method, this allowed us to make confident conclusions about the similarities of the complex bacterial communities in the 17 different samples.

Osborne, Catherine A.; Rees, Gavin N.; Bernstein, Yaniv; Janssen, Peter H.

2006-01-01

233

High-Frequency Genetic Recombination and Reactivation of Orthopoxviruses from DNA Fragments Transfected into Leporipoxvirus-Infected Cells  

Microsoft Academic Search

and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green

Xiao-Dan Yao; David H. Evans

2003-01-01

234

Dependence on radiation quality of DNA fragmentation spectra  

NASA Astrophysics Data System (ADS)

Energy deposition by radiation initially gives rise to cellular critical lesions such as DNA doublestrand breaks (DSB), that later lead to the formation of relevant biological endpoints. Studies on fragment size distributions induced by radiations of various qualities can be of great help in linking the characteristics of radiation to cellular endpoints, providing information for understanding the main mechanisms of cell damage. Here we are concerned with the damage induced by heavy charged particles; this issue is very important in the field of radioprotection of astronauts participating in long term space missions, besides being relevant also in other fields, like hadrontherapy. Galactic Cosmic Rays contain a large component of high-LET particles (HZE), e.g. helium and carbon ions, as well as highcharge particles such as iron ions. These particles are characterized by complex track structures with energy depositions not only along the path of the primary particle, but also at relatively large distance form the path, due to the presence of high energy secondary electrons. In this work we have simulated the irradiation of human fibroblasts with ?-rays, protons, helium, carbon and iron ions at a fixed dose with the biophysical Monte Carlo code PARTRAC,and calculated the induction of DSB. The PARTRAC code includes accurate representation of the chromatin geometry and of the physical and physico-chemical processes associated with the energy deposition by radiation. The results of a first validation of the code have been reported in A. Campa et al. (2005) and D. Alloni et al. (2007a, 2007b). DNA fragment spectra were calculated based on the DSB induction patterns and compared in particular for particles of the same specific energy and for particles of the same LET. Special emphasis has been directed to the calculation of very small fragments (< 1 kbp) that are not detectable by the most common experimental techniques and that can significantly influence the RBE (Relative Biological Effectiveness) of high LET radiation. This work was partially supported by EU ("RISC-RAD" project, Contract no. FI6R-CT 2003- 508842, and "NOTE" project, Contract no. FI6R-036465) and ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W.Friedland, S. Gerardi, S. Molinelli, A. Ottolenghi, H. G. Paretzke, G. Simone and M. A. Tabocchini. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat.Biol. 81, 841-854 (2005). D. Alloni, F. Ballarini, M. Belli, A. Campa, G. Esposito, W. Friedland, M.Liotta, A. Ottolenghi and H. G. Paretzke. Modeling of DNA fragmentation induced in human fibroblasts by 56 Fe ions. Adv. Space Res. 40, 1401-1407 (2007a). D. Alloni, F. Antonelli, F. Ballarini, M. Belli, A. Campa, V. Dini, G.Esposito, W. Friedland, M. Liotta, A. Ottolenghi, H. G. Paretzke, G. Simone, E. Sorrentino and M. A. Tabocchini. Small DNA fragments induced in human fibroblasts by 56 Fe ions: experimental data and MC simulations. Proc. "Ion Beams in biology and medicine", Heidelberg, 26-29 September 2007, edited by J. Debus, K. Henrichs, G. Kraft, p. 164 (2207b).

Campa, Alessandro; Ottolenghi, Andrea; Alloni, Daniele; Ballarini, Francesca; Belli, Mauro; Esposito, Giuseppe; Facoetti, Angelica; Friedland, Werner; Liotta, Marco; Paretzke, Herwig

235

Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5.  

PubMed Central

We have established a library of hamster cells transformed by adenovirus 5 DNA fragments comprising all (XhoI-C, 0 to 16 map units) or only a part (HindIII-G, 0 to 7.8 map units) of early region 1 (E1: 0 to 11.2 map units). These lines have been analyzed in terms of content of viral DNA, expression of E1 antigens, and capacity to induce tumors in hamsters. All cells tested were found to express up to eight proteins encoded within E1A (0 to 4.5 map units) with apparent molecular weights between 52,000 (52K) and 25K. Both G and C fragment-transformed lines expressed a 19K antigen encoded within E1B (4.5 to 11.2 map units), whereas an E1B 58K protein was detected in C fragment-transformed, but not G-fragment-transformed, lines. No clear distinction could be drawn between cells transformed by HindIII-G and by XhoI-C in terms of morphology or tumorigenicity, suggesting that the E1B 58K antigen plays no major role in the maintenance of oncogenic transformation, although possible involvement of truncated forms of 58K cannot be ruled out. Sera were collected from tumor-bearing animals and examined for ability to immunoprecipitate proteins from infected cells. The relative avidity of sera for different proteins was characteristic of the cell line used for tumor induction, and the specificity generally reflected the array of viral proteins expressed by the corresponding transformed cells. However, one notable observation was that even though all transformed lines examined expressed antigens encoded by both the 1.1- and 0.9-kilobase mRNAs transcribed from E1A, tumor sera made against these lines only precipitated products of the 1.1-kilobase message. Thus, two families of E1A proteins, highly related in terms of primary amino acid sequence, appear to be immunologically quite distinct. Images

Rowe, D T; Branton, P E; Yee, S P; Bacchetti, S; Graham, F L

1984-01-01

236

Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men  

Microsoft Academic Search

Objective: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters.Design: Prospective, observational study.Setting: University infertility clinic.Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy.Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF.Main

Armand Zini; Ryszard Bielecki; Donna Phang; Maria Teresa Zenzes

2001-01-01

237

Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases  

PubMed Central

In T4 bacteriophage, 5-hydroxymethylcytosine (5hmC) is incorporated into DNA during replication. In response, bacteria may have developed modification-dependent type IV restriction enzymes to defend the cell from T4-like infection. PvuRts1I was the first identified restriction enzyme to exhibit specificity toward hmC over 5-methylcytosine (5mC) and cytosine. By using PvuRts1I as the original member, we identified and characterized a number of homologous proteins. Most enzymes exhibited similar cutting properties to PvuRts1I, creating a double-stranded cleavage on the 3? side of the modified cytosine. In addition, for efficient cutting, the enzymes require two cytosines 21–22-nt apart and on opposite strands where one cytosine must be modified. Interestingly, the specificity determination unveiled a new layer of complexity where the enzymes not only have specificity for 5-?-glucosylated hmC (5?ghmC) but also 5-?-glucosylated hmC (5?ghmC). In some cases, the enzymes are inhibited by 5?ghmC, whereas in others they are inhibited by 5?ghmC. These observations indicate that the position of the sugar ring relative to the base is a determining factor in the substrate specificity of the PvuRts1I homologues. Lastly, we envision that the unique properties of select PvuRts1I homologues will permit their use as an additive or alternative tool to map the hydroxymethylome.

Borgaro, Janine G.; Zhu, Zhenyu

2013-01-01

238

PCR-Restriction Fragment Length Polymorphism Analysis of the Phospholipase B (PLB1) Gene for Subtyping of Cryptococcus neoformans Isolates  

Microsoft Academic Search

Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report describes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) as a simple tool to differentiate between C. neoformans subgroups. A PLB1 fragment, 1,970 bp, was amplified and digested with either Ava

G. Nicolas Latouche; Matthew Huynh; Tania C. Sorrell; Wieland Meyer

2003-01-01

239

Ca2 antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen  

Microsoft Academic Search

Ca2 accumulates in the nucleus and DNA undergoes enzymatic cleavage into internucleosome- length fragments before acetaminophen and dimethyl- nitrosamine produce hepatic necrosis in vivo and toxic cell death in vitro. However, Ca2-endonudease fragmen- tation of DNA is characteristic of apoptosis, a type of cell death considered biochemically and functionally distinct from toxic cell death. The present studies investigate DNA fragmentation

SIDHARTHA D. RAY; LISA M KAMENDULIS; MARK W. GURULE; ROBERT D. YORKIN; GEORGE B. CORCORAN

1993-01-01

240

Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA ®)  

Microsoft Academic Search

Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope.SCSA measurements of animal or

Donald P.. Evenson; Regina Wixon

2005-01-01

241

Thermodynamic DNA Looping by a Two-Site Restriction Endonuclease Studied using Optical Tweezers  

Microsoft Academic Search

Many enzyme-DNA interactions involve multimeric protein complexes that bind at two distant sites such that the DNA is looped. An example is the type IIe restriction enzyme Sau3AI, which requires two recognition sites to cleave the DNA. Here we study this process at the single DNA level using force measuring optical tweezers. We characterize cleavage rates of single DNA molecules

Gregory J. Gemmen

2005-01-01

242

Restriction enzyme cleavage maps of the DNA of two cauliflower mosaic virus isolates.  

PubMed

To provide a physical basis for the functional mapping of the cauliflower mosaic virus (CaMV) genome, restriction enzyme cleavage maps of the DNA of CaMV isolates NY8153 and CM4-184-OS (a variant of CM4-184) were constructed. CM4-184-OS DNA contained five EcoRI, eight HindIII six BglIII, and two BamHI cleavage sites, while NY8153 DNA contained one additional HindIII site and one fewer EcoRI site. Fragments were ordered by examination of multienzyme and partial digests. The resulting maps revealed that the CM4-184-OS genome was 5.5% smaller than that of NY8153, because CM4-184-OS was deleted for 463 +/- 10 base pairs including the HindIII site at 0.427 map unit (relative to a common EcoR1 site) and the S1 site at 0.45 map unit on NY8153 DNA. Since the deleted region did not include the two BamHI sites, this variant of the CM4-184 isolate is different from the John Innes variant of CM4-184 (R. Hull, and S. H. Howell, Virology 86, 482-493, 1978) which is deleted for a different, but overlapping region of the CaMV genome. PMID:18631651

Gardner, C O; Melcher, U; Shockey, M W; Essenberg, R C

1980-05-01

243

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites  

Microsoft Academic Search

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was

Tomomasa Watanabe; Yukimasa Hayashi; Reiji Semba; Nobuaki Ogasawara

1985-01-01

244

Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.  

PubMed Central

Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. Moreover, the techniques we have described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems. Images

Joyce, C M; Grindley, N D

1983-01-01

245

Temporal Patterns of Nucleotide Misincorporations and DNA Fragmentation in Ancient DNA  

PubMed Central

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5?-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.

Sawyer, Susanna; Krause, Johannes; Guschanski, Katerina; Savolainen, Vincent; Paabo, Svante

2012-01-01

246

Characterization of cytomegalovirus isolates from patients with AIDS by DNA restriction analysis.  

PubMed Central

Thirty-seven isolates of cytomegalovirus (CMV) were obtained from a group of 20 promiscuous homosexual men, either suffering from the acquired immunodeficiency syndrome (AIDS) at the time of CMV isolation, or who developed AIDS subsequently. The isolates of CMV were characterized by the method of DNA restriction analysis. All epidemiologically unrelated strains of CMV exhibited different fragment migration patterns and no one strain appeared to be associated with AIDS or any particular disease pattern in these patients. Sequential isolates of CMV were obtained from nine patients in the study group either from different sites at the same time or from the same site on different dates. In the case of seven of the men, viruses with minor differences in restriction profile were obtained, possibly representing sub-populations of an endogenous strain of CMV. In two of the patients, reinfection with different strains was apparent. We conclude that reinfections with CMV in AIDS patients can occur, but the isolation of strains exhibiting major differences in genome structure seen by restriction enzyme analysis was uncommon. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

Taylor, D. L.; Taylor-Robinson, D.; Jeffries, D. J.; Tyms, A. S.

1988-01-01

247

Comparison of bacterial communities in the throat swabs from healthy subjects and pharyngitis patients by terminal restriction fragment length polymorphism.  

PubMed

Terminal restriction fragment length polymorphism (T-RFLP) analysis was applied to characterize bacterial flora present in the throats of healthy subjects and pharyngitis patients. The 16S rRNA genes of bacteria present in throat metagenome were amplified by PCR with 6-carboxy-fluorescein (6-FAM)-labeled universal forward primer (27 F) and a universal reverse primer (1513R). The 16S rDNAs were digested with restriction enzymes with 4-bp recognition sites (MspI or RsaI) and analyzed by using an automated DNA sequencer. T-RFLP patterns were numerically analyzed using computer programs. From analysis of the throat bacterial community, patterns derived from MspI and RsaI digested samples of healthy subjects and pharyngitis patients were grouped into different clusters, though RsaI digested samples showed some uncertainty. Pharyngitis throats generated an average species richness of 9 [±2.1 (SD)] and 10 (±2.9) for MspI and RsaI digests, respectively, whereas healthy throats generated 6.3 (±1.2) and 6.1 (±1.5) in MspI and RsaI digests, respectively. These results suggest that samples from pharyngitis patients contain an unexpected diversity of causative bacteria. The pharyngitis throats were colonized with a rich diversity of bacterial species than that of healthy throats. Using T-RFLP, we are able to detect a model bacterium, Streptococcus pyogenes SF370, and T-RF patterns were consistent with the Streptococcal T-RFLP patterns. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of throat bacterial flora and rapid comparison of the community structure between subjects with and without pharyngitis. PMID:22322827

Balaji, Kannan; Thenmozhi, Ramalingam; Sundaravadivel, Marimuthu; Pandian, Shunmugiah Karutha

2012-02-10

248

IS6110 Restriction Fragment Length Polymorphism Typing of Drug-resistant Mycobacterium tuberculosis Strains from Northeast South Africa  

PubMed Central

Tuberculosis (TB) remains a deadly infectious disease affecting millions of people worldwide; 95% of TB cases, with 98% of death occur in developing countries. The situation in South Africa merits special attention. A total of 21,913 sputum specimens of suspected TB patients from three provinces of South Africa routinely submitted to the TB laboratory of Dr. George Mukhari (DGM) Hospital were assayed for Mycobacterium tuberculosis (MTB) growth and antibiotic susceptibility. The genetic diversity of 338 resistant strains were also studied. DNA isolated from the strains were restricted with Pvu II, transferred on to a nylon membrane and hybridized with a PCR-amplified horseradish peroxidase 245 bp IS6110 probe. Of the 338 resistant strains, 2.09% had less than 5 bands of IS6110, and 98% had 5 or more bands. Unique restriction fragment length polymorphism (RFLP) patterns were observed in 84.3% of the strains, showing their epidemiological independence, and 15.7% were grouped into 22 clusters. Thirty-two strains (61.5%) from the 52 that clustered were from Mpumalanga, 16/52 (30.8%) from Gauteng, and 4/52 (9.6%) from Limpopo province. Clustering was not associated with age. However, strains from male patients in Mpumalanga were more likely to be clustered than strains from male patients in Limpopo and/or Gauteng province. The minimum estimate for the proportion of resistant TB that was due to transmission is 9.06% (52-22=30/331). Our results indicate that transmission of drug-resistant strains may contribute substantially to the emergence of drug-resistant tuberculosis in South Africa.

Green, Ezekiel; Obi, Lawrence C.; Okoh, Anthony I.; Nchabeleng, Maphoshane; Villiers, Babsie E. De; Letsoalo, Tomas; Hoosen, Anwar A.; Bessong, Pascal O.

2013-01-01

249

IS6110 restriction fragment length polymorphism typing of drug-resistant Mycobacterium tuberculosis strains from northeast South Africa.  

PubMed

Tuberculosis (TB) remains a deadly infectious disease affecting millions of people worldwide; 95% of TB cases, with 98% of death occur in developing countries. The situation in South Africa merits special attention. A total of 21,913 sputum specimens of suspected TB patients from three provinces of South Africa routinely submitted to the TB laboratory of Dr. George Mukhari (DGM) Hospital were assayed for Mycobacterium tuberculosis (MTB) growth and antibiotic susceptibility. The genetic diversity of 338 resistant strains were also studied. DNA isolated from the strains were restricted with Pvu II, transferred on to a nylon membrane and hybridized with a PCR-amplified horseradish peroxidase 245 bp IS6110 probe. Of the 338 resistant strains, 2.09% had less than 5 bands of IS6110, and 98% had 5 or more bands. Unique restriction fragment length polymorphism (RFLP) patterns were observed in 84.3% of the strains, showing their epidemiological independence, and 15.7% were grouped into 22 clusters. Thirty-two strains (61.5%) from the 52 that clustered were from Mpumalanga, 16/52 (30.8%) from Gauteng, and 4/52 (9.6%) from Limpopo province. Clustering was not associated with age. However, strains from male patients in Mpumalanga were more likely to be clustered than strains from male patients in Limpopo and/or Gauteng province. The minimum estimate for the proportion of resistant TB that was due to transmission is 9.06% (52-22 = 30/331). Our results indicate that transmission of drug-resistant strains may contribute substantially to the emergence of drug-resistant tuberculosis in South Africa. PMID:23617199

Green, Ezekiel; Obi, Lawrence C; Okoh, Anthony I; Nchabeleng, Maphoshane; de Villiers, Babsie E; Letsoalo, Tomas; Hoosen, Anwar A; Bessong, Pascal O; Ndip, Roland N

2013-03-01

250

RegionSpecific Interrelations between Apoptotic Proteins Expression and DNA Fragmentation in the Neonatal Rat Brain  

Microsoft Academic Search

DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL\\/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and

Petr N. Menshanov; Anita V. Bannova; Nikolay N. Dygalo

2006-01-01

251

Comparison of bacterial communities in New England Sphagnum bogs using terminal restriction fragment length polymorphism (T-RFLP).  

PubMed

Wetlands are major sources of carbon dioxide, methane, and other greenhouse gases released during microbial degradation. Despite the fact that decomposition is mainly driven by bacteria and fungi, little is known about the taxonomic diversity of bacterial communities in wetlands, particularly Sphagnum bogs. To explore bacterial community composition, 24 bogs in Vermont and Massachusetts were censused for bacterial diversity at the surface (oxic) and 1 m (anoxic) regions. Bacterial diversity was characterized by a terminal restriction fragment length (T-RFLP) fingerprinting technique and a cloning strategy that targeted the 16S rRNA gene. T-RFLP analysis revealed a high level of diversity, and a canonical correspondence analysis demonstrated marked similarity among bogs, but consistent differences between surface and subsurface assemblages. 16S rDNA sequences derived from one of the sites showed high numbers of clones belonging to the Deltaproteobacteria group. Several other phyla were represented, as well as two Candidate Division-level taxonomic groups. These data suggest that bog microbial communities are complex, possibly stratified, and similar among multiple sites. PMID:16729225

Morales, Sergio E; Mouser, Paula J; Ward, Naomi; Hudman, Stephen P; Gotelli, Nicholas J; Ross, Donald S; Lewis, Thomas A

2006-05-31

252

Restriction Fragment Length Polymorphism Analysis of Some Flagellin Genes of Salmonella enterica  

PubMed Central

Salmonellae often have the ability to express two different flagellar antigen specificities (phase 1 and phase 2). At the cell level, only one flagellar phase is expressed at a time. Two genes, fliC, encoding phase-1 flagellin, and fljB, encoding phase-2 flagellin, are alternatively expressed. Flagellin genes from 264 serovars of Salmonella enterica were amplified by two phase-specific PCR systems. Amplification products were subjected to restriction fragment length polymorphism (RFLP) analysis by using endonucleases HhaI and HphI. RFLP with HhaI and HphI yielded 64 and 42 different restriction profiles, respectively, among 329 flagellin genes coding for 26 antigens. The phase-1 gene showed 46 patterns with HhaI and 30 patterns with HphI. The phase-2 gene showed 23 patterns with HhaI and 17 patterns with HphI. When the data from both enzymes were combined, 116 patterns were obtained: 74 for fliC, 47 for fljB, and 5 shared by both genes. Of these combined patterns, 80% were specifically associated with one flagellar antigen and 20% were associated with more than one antigen. Each flagellar antigen was divided into 2 to 18 different combined patterns. In the sample of strains used, determination of the phase-1 and phase-2 flagellin gene RFLP, added to the knowledge of the O antigen, allowed identification of all diphasic serovars. Overall, the diversity uncovered by flagellin gene RFLP did not precisely match that evidenced by flagellar agglutination.

Dauga, Catherine; Zabrovskaia, Anna; Grimont, Patrick A. D.

1998-01-01

253

Application of small fragment restriction endonuclease analysis (SF REA) to the epidemiological f i n ge rp r i n t i n g of Staph y\\/ococcus aureus  

Microsoft Academic Search

Summary. Total cell DNA of 14 isolates of Staphylococcus aureus from patients of an intensive care unit (ICU) and 180 unrelated strains was examined by restriction endonuclease analysis (REA). EcoRI-generated DNA fragments were either subjected to conventional REA on agarose gels and stained with ethidium bromide or separated by polyacrylamide gel electrophoresis and visualised by silver staining (SF-REA). Both methods

R. HAERTL; G. BANDLOW

1990-01-01

254

Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences.  

PubMed Central

We have established a sensitive, monoclonal antibody (Mab)-based procedure permitting the selective enrichment of sequences containing the miscoding alkylation product O6-ethylguanine (O6-EtGua) from mammalian DNA. H5 rat hepatoma cells were reacted with the N-nitroso carcinogen N-ethyl-N-nitrosourea in vitro, to give overall levels of greater than or equal to 25 O6-EtGua residues per diploid genome (corresponding to O6-EtGua/guanine molar ratios of greater than or equal to 10(-8). For analysis, enzymatically restricted DNA from these cells is incubated with an antibody specific for O6-ethyl-2'-deoxyguanosine, the resulting Mab-DNA complexes are separated from (O6-EtGua)-free fragments by filtration through a nitrocellulose (NC) membrane, and the DNA is recovered from the filter-bound complexes quantitatively. The efficiency of Mab binding to DNA fragments containing O6-EtGua is constant over a range of O6-EtGua/guanine molar ratios between 10(-5) and 10(-8). (O6-EtGua)-containing restriction fragments encompassing known gene sequences (e.g., the immunoglobulin E heavy chain gene of H5 rat hepatoma cells used as a model in this study) are subsequently amplified by PCR and quantified by slot-blot hybridisation. The content and distribution of a specific carcinogen-DNA adduct in defined sequences of genomic DNA can thus be analyzed as well as the kinetics of intragenomic (toposelective) repair of any DNA lesion for which a suitable Mab is available.

Hochleitner, K; Thomale, J; Nikitin AYu; Rajewsky, M F

1991-01-01

255

Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales  

PubMed Central

A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation.

Redecker, D.; Thierfelder, H.; Walker, C.; Werner, D.

1997-01-01

256

Restriction fragment length polymorphism within the class I gene loci of the equine major histocompatibility complex  

SciTech Connect

Fourteen standard bred horses were serotyped as homozygous for 1 of 6 Equine Leukocyte Antigen (ELA) specificities. DNA was purified from peripheral leukocytes and digested with Hind III or Pvu II. Southern blot hybridization analysis was carried out using a /sup 32/P-labeled mouse cDNA probe (PH2IIa) specific for class I MHC genes. Both enzymes generated blots that contained a large number of bands (23 to 30) per horse. Significant polymorphism existed among most fragment sizes, while a dozen highly conserved band sizes suggested the presence of Qa/tla - like genes. Only 2 animals (both W6's) showed identical band patterns. Polymorphism was greatest between horses of different serotypes and was significantly decreased within serotypes. Unique bands were present on both blots for both W1's and W6's and may account for the serologic specificity seen in ELA W1 and W6 horses. This study is consistent with the findings in other higher vertebrates and implies that the MHC of the horse includes a highly polymorphic class I multigene family.

Alexander, A.J.; Bailey, E.; Woodward, J.G.

1986-03-05

257

A Time-Efficient and User-Friendly Method for Plasmid DNA Restriction Analysis.  

ERIC Educational Resources Information Center

|Describes an experiment in which plasmid DNA is digested with restriction enzymes that cleave the plasmid either once or twice. The DNA is stained, loaded on a gel, electrophoresed, and viewed under normal laboratory conditions during electrophoresis. (DDR)|

LaBanca, Frank; Berg, Claire M.

1998-01-01

258

Restriction fragment length polymorphism analysis of Cryptococcus neoformans isolates from environmental (pigeon excreta) and clinical sources in New York City.  

PubMed Central

Restriction fragment length polymorphism analysis of environmental (pigeon excreta) and clinical Cryptococcus neoformans var. neoformans isolates in a limited geographic area distinguished 6 strains among 8 environmental isolates and 12 strains among 17 clinical isolates. Clusters of patients with three strains types accounted for 47% of clinical isolates. Despite this diversity, two strains were shared by environmental and clinical isolates. Images

Currie, B P; Freundlich, L F; Casadevall, A

1994-01-01

259

Allozyme and Restriction Fragment Length Polymorphism Analyses Confirm Entomophaga maimaiga Responsible for 1989 Epizootics in North American Gypsy Moth Populations  

Microsoft Academic Search

In 1989, populations of North American gypsy moth, Lymantria dispar, in seven contiguous northeastern states were severely reduced by a fungal pathogen. Based on morphology, development, and pathology, this organism appeared to be Entomophaga maimaiga. We have now used allozyme and restriction fragment length polymorphism analyses to confirm this identification. Previously, this mycopathogen had been reported only from gypsy moth

Ann E. Hajek; Richard A. Humber; Joseph S. Elkinton; Bernie May; Scott R. A. Walsh; Julie C. Silver

1990-01-01

260

Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis  

Microsoft Academic Search

The hepatitis D virus (HDV) genotypes in 46 HDV- infected patients and 12 prostitutes were screened with XhoI restriction fragment length polymorphism (RFLP) analysis of reverse transcription PCR pro- ducts of viral genomes and verified by phylogenetic analysis. The amplificates of three (6-5%) patients and two (17%) prostitutes showed a novel RFLP pattern different from those of the three known

Jaw-Ching Wu; Tzen-Yuh Chiang; I-Jane Sheen

1998-01-01

261

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosome 12q  

Microsoft Academic Search

Human gene mapping would be greatly facilitated if marker loci with sufficient polymorphism information content were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have used a rapid method for screening random cosmids to identify those homologous to genomic regions especially rich in restriction fragment length polymophisms (Litt and White

N. E. Buroker; R. E. Magenis; K. Weliky; G. Bruns; M. Litt

1986-01-01

262

Very Efficient Template\\/Primer-Independent DNA Synthesis by Thermophilic DNA Polymerase in the Presence of a Thermophilic Restriction Endonuclease  

Microsoft Academic Search

We have found that, in the presence of a thermophilic restriction endonuclease, thermophilic DNA polymerase efficiently synthesizes and amplifies DNA in the absence of any added template and primer nucleic acid under isothermal conditions. More than 10 Ěg of DNA can be synthesized by 1 unit of DNA polymerase in 1 h, and the reaction proceeds until available dNTPs are

Xingguo Liang; Kari Jensen; Maxim D. Frank-Kamenetskii

2004-01-01

263

Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms  

SciTech Connect

A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine.

Nobile, C.; Romeo, G.

1988-10-01

264

Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.  

PubMed Central

Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes.

McClelland, M; Nelson, M; Raschke, E

1994-01-01

265

Autonomous replication of human chromosomal DNA fragments in human cells.  

PubMed Central

We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments. Images

Masukata, H; Satoh, H; Obuse, C; Okazaki, T

1993-01-01

266

GB Virus C/Hepatitis G Virus Groups and Subgroups: Classification by a Restriction Fragment Length Polymorphism Method Based on Phylogenetic Analysis of the 5? Untranslated Region  

PubMed Central

A phylogenetic tree based on 150 5? untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that HinfI and either AatII or AciI could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out.

Quarleri, J. F.; Mathet, V. L.; Feld, M.; Ferrario, D.; della Latta, M. P.; Verdun, R.; Sanchez, D. O.; Oubina, J. R.

1999-01-01

267

Identification of raw and heat-processed meats from game bird species by polymerase chain reaction-restriction fragment length polymorphism of the mitochondrial D-loop region.  

PubMed

Polymerase chain reaction-RFLP analysis has been applied to the identification of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), red-legged partridge (Alectoris rufa), chukar partridge (Alectoris chukar), guinea fowl (Numida meleagris), capercaillie (Tetrao urogallus), Eurasian woodcock (Scolopax rusticola), and woodpigeon (Columba palumbus). Polymerase chain reaction amplification was carried out using a set of primers flanking a conserved region of approximately 310 bp from the mitochondrial D-loop region. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of HinfI, MboII, and Hpy188III endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all game bird species analyzed. Consistent results were obtained with both raw and heat-processed meats. PMID:19211540

Rojas, M; González, I; Fajardo, V; Martín, I; Hernández, P E; García, T; Martín, R

2009-03-01

268

Identification of Cryptosporidium spp. Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay  

PubMed Central

We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.

Nichols, R. A. B.; Campbell, B. M.; Smith, H. V.

2003-01-01

269

Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms (MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analyses  

PubMed Central

DNA was isolated from clover proliferation (CP) mycoplasmalike organism (MLO)-diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned into pSP6 plasmid vectors. CP MLO-specific recombinant DNA clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among CP MLO and other MLOs, including potato witches'-broom (PWB) MLO. Results from dot hybridization analyses indicated that both a Maryland strain of aster yellows and a California strain of aster yellows are distantly related to CP MLO. Elm yellows, paulownia witches'-broom, peanut witches'-broom, loofah witches'-broom, and sweet potato witches'-broom may be very distantly related, if at all, to CP MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover phyllody MLO, beet leafhopper-transmitted virescence MLO, and ash yellows MLO are related to CP MLO, but PWB MLO is the most closely related. Similarity coefficients derived from restriction fragment length polymorphism analyses revealed that PWB and CP MLOs are closely related strains and thus provided direct evidence of their relatedness in contrast to reliance solely on biological characterization. Images

Lee, Ing-Ming; Davis, Robert E.; Hiruki, Chuji

1991-01-01

270

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay.  

PubMed

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI-TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders. PMID:21262192

Cha, Yoon Seok; Choi, Suh Hee; Lee, Joo-Hyoung; Shin, Soo-Kyung; Lee, Seung Hwan; Lee, Soong Deok; Kim, Soo-Ok; Hong, Sun Pyo

2011-01-22

271

Establishment and characterization of hamster cell line transformed by restriction endonuclease fragments of Adenovirus 5  

SciTech Connect

The authors have established a library of hamster cells transformed by adenvirus 5 DNA fragments comprising all or only a part of early region 1. These lines have been analyzed in terms of content of viral DNA, expression of E1 antigens, and capacity to induce tumors in hamsters. All cells tested were found to express up to eight proteins encoded within E1A (0 to 4.5 map units) with apparent molecular weights between 52,000 (52K) and 25K. No clear distinction could be drawn between cells transformed by HindIII-G and by Xhol-I-C in terms of morphology or tumorigenicity, suggesting that the E1B 58K antigen plays no major role in the maintenance of oncogenic transformation, although possible involvement of truncated forms of 58K cannot be ruled out. Sera were collected from tumor-bearing animals and examined for ability to immunoprecipitate proteins from infected cells. The relative avidity of sera for different proteins was characteristic of the cell line used for tumor induction, and the specificity generally reflected the array of viral proteins expressed by the corresponding transformed cells. However, one notable observation was that even though examined expressed antigens enoded by both the 1.1- and 0.9-kilobase mRNAs transcribed from E1A, tumor sera made against these lines only precipitated products of the 1.1-kilobase message. Thus, two families of E1A proteins, highly related in terms of primary amino acid sequence, appear to be immunologically quite distinct.

Rowe, D.T.; Branton, P.E.; Yee, S.P.; Bacchetti, S.; Graham, F.L.

1984-01-01

272

A Mini-Library of Sequenced Human DNA Fragments: Linking Bench Experiments with Informatics  

ERIC Educational Resources Information Center

|We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating "wet-lab" and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in "Escherichia coli" remains a fundamental…

Dalgleish, Raymond; Shanks, Morag E.; Monger, Karen; Butler, Nicola J.

2012-01-01

273

PCR-based restriction fragment length polymorphism and haplotype of the most common mutation L176F in the beta-glucuronidase gene.  

PubMed

Mucopolysaccharidosis type VII or Sly syndrome is an autosomal recessive disorder of glycosaminoglycan storage leading to variable clinical symptoms, such as hepatosplenomegaly, bone deformities, hearing loss, corneal opacities, mental retardation, and hydrops fetalis in affected individuals. The disease is caused by approximately 40 different mutations in the beta-glucuronidase gene. Detection of the most common mutation L176F by single-strand conformation polymorphism (SSCP) was not always successful. Although DNA sequencing followed by PCR amplification can easily detect this mutation, accessibility to a DNA sequencer or useful reagents in the sequencing procedure is not readily available in many countries. A PCR-based restriction fragment length polymorphism (RFLP) developed in this report would allow rapid and easier detection of this mutation for screening new patients or neonates of heterozygous parents. Analysis of intragenic polymorphic sites in the L176F patients identified two distinct alleles; the predominant one probably originated in Spain. PMID:17394395

Islam, M Rafiq; Shah, Gul N; Sly, William S

2007-01-01

274

[Fibronectin fragmentation unmasks the activity stimulating DNA and RNA biosynthesis in granulation tissue cells in vitro].  

PubMed

Human blood plasma fibronectin decreased slightly the incorporation of precursors into nucleic acids of granulation tissue culture cells. A slight fragmentation of fibronectin, where the fragments with 180-200 kD molecular mass were developed, led to occurrence of the activity 2-fold stimulating the DNA synthesis. After more effective proteolysis using plasmin and trypsin the stimulating effect of fibronectin fragments on synthesis of nucleic acids maintained and constituted 165 +/- 12% and 127 +/- 7% for DNA and RNA, respectively. PMID:2437701

Zlatopol'ski?, A D; Za?denberg, M A; Berman, A E; Mazurov, V I; Karelin, A A

275

Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism.  

PubMed

Terminal restriction fragment length polymorphism (T-RFLP) can be used to assess how land use management changes the dominant members of bacterial communities. We compared T-RFLP profiles obtained via amplification with forward primers (27, 63F) each coupled with the fluorescently labeled reverse primer (1392R) and multiple restriction enzymes to determine the best combination for interrogating soil bacterial populations in an agricultural soil used for potato production. Both primer pairs provide nearly universal recognition of a 1,400-bp sequence of the bacterial domain in the V(1)-V(3) region of the 16S ribosomal RNA (rRNA) gene relative to known sequences. Labeling the reverse primer allowed for direct comparison of each forward primer and the terminal restriction fragments' relative migration units obtained with each primer pair and restriction enzyme. Redundancy analysis (RDA) and nested multivariate analysis of variance (MANOVA) were used to assess the effects of primer pair and choice of restriction enzyme on the measured relative migration units. Our research indicates that the 63F-1392R amplimer pair provides a more complete description with respect to the bacterial communities present in this potato (Solanum tuberosum L.)-barley (Hordeum vulgare L.) rotation over seeded to crimson clover (Trifolium praense L.). Domain-specific 16S rRNA gene primers are rigorously tested to determine their ability to amplify across a target region of the gene. Yet, variability within or between T-RFLP profiles can result from factors independent of the primer pair. Therefore, researchers should use RDA and MANOVA analyses to evaluate the effects that additional laboratory and environmental variables have on bacterial diversity. PMID:21667276

Fortuna, Ann-Marie; Marsh, Terence L; Honeycutt, C Wayne; Halteman, William A

2011-06-12

276

Amplified ribosomal DNA restriction analysis in the differentiation of related species of mycobacteria  

Microsoft Academic Search

This study explores the potential of the amplified ribosomal DNA restriction analysis (ARDRA) for intra- and interspecies identification of the genus Mycobacteria. A set of primers was used to amplify part of the 16S and 23S rDNA as well as the 16S–23S rDNA spacer from 121 isolates belonging to 13 different mycobacterial species. Restriction analysis was carried out with five

Mekonnen Kurabachew; Řivind Enger; Ruth-Anne Sandaa; Eshetu Lemma; Bjarne Bjorvatn

2003-01-01

277

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns  

Microsoft Academic Search

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc

Tomomasa Watanabe; Yukimasa Hayashi; Nobuaki Ogasawara; Takeshi Tomoita

1985-01-01

278

Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay  

Microsoft Academic Search

BACKGROUND: Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused

María Tamayo; Rebeca Santiso; Jaime Gosalvez; Germán Bou; José Luis Fernández

2009-01-01

279

Chromosomal localization of a highly repeated Eco RI DNA fragment in Megoura viciae (Homoptera, Aphididae) by nick translation and fluorescence in situ hybridization  

Microsoft Academic Search

To investigate the genome of the aphidMegoura viciae at molecular level, we have studied total DNA by agarose gel electrophoresis after cleavage with different restriction endonucleases.EcoRI digestion produced a highly repeated DNA fragment, about 600 bp long. The contribution of thisEcoRI element to the total genome ofM. viciae was estimated at about 6% by means of densitometric scanning of agarose

Davide Bizzaro; Gian Carlo Manicardi; Umberto Bianchi

1996-01-01

280

Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.  

PubMed Central

It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment.

de Vroom, E; Roelen, H C; Saris, C P; Budding, T N; van der Marel, G A; van Boom, J H

1988-01-01

281

Tailoring the activity of restriction endonuclease PleI by PNA-induced DNA looping  

Microsoft Academic Search

DNA looping is one of the key factors allowing proteins bound to different DNA sites to signal one another via direct contacts. We demonstrate that DNA looping can be generated in an arbitrary chosen site by sequence-directed targeting of double-stranded DNA with pseudocomplementary peptide-nucleic acids (pcPNAs). We designed pcPNAs to mask the DNA from cleavage by type IIs restriction enzyme

Ekaterina Protozanova; Vadim V. Demidov; Viatcheslav Soldatenkov; Sergey Chasovskikh; Maxim D. Frank-Kamenetskii

2002-01-01

282

Development of a PCR-Restriction Fragment Length Polymorphism Assay Using the Nucleotide Sequence of the Helicobacter hepaticus Urease Structural Genes ureAB  

PubMed Central

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.

Shen, Zeli; Schauer, David B.; Mobley, Harry L. T.; Fox, James G.

1998-01-01

283

Forensic identification of ungulate species using restriction digests of PCR-amplified mitochondrial DNA.  

PubMed

A survey of mitochondrial D-loop variation in 15 species of ungulates was conducted via amplification by the polymerase chain reaction followed by restriction fragment length polymorphism analysis. This survey included moose (Alces alces), caribou (Rangifer tarandus), mule deer (Odocoileus hemionus hemionus), black-tailed deer (O. h. columbianus), white-tailed deer (O. virginianus), waipiti (Cervus elaphus), pronghorn antelope (Antilocapra americana), bighorn sheep (Ovis canadensis), Stone's sheep (O. dalli), domestic sheep (O. aries), moulflon sheep (O. musimon), mountain goat (Oreamnos americanus), domestic goat (Capra hircus), domestic cattle (Bos taurus), and bison (Bison bison). The results of this preliminary survey indicate that there may be sufficient species specific variation in the D-loop region of the mitochondrial genome of the ungulate species examined here, with the exception of deer (Odocoileus) species, to establish the species origin of the mitochondrial haplotypes of this group. The Odocoileus species are known to hybridize and sharing of mtDNA haplotypes was observed. The chelex DNA extraction technique was successfully used on small blood stains. PMID:8522926

Murray, B W; McClymont, R A; Strobeck, C

1995-11-01

284

Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA  

Microsoft Academic Search

Fragments of phage T7 DNA have been cloned in Escherichia coli by using the plasmid pMB9. Such cloned fragments are able to recombine with infecting phages, thus providing a means to integrate the physical and genetic maps of T7 DNA. Approximately 65% of the T7 DNA molecule has been found in clones so far, and analysis of these clones has

J. L. Campbell; C. C. Richardson; F. W. Studier

1978-01-01

285

Routine Use of PCR-Restriction Fragment Length Polymorphism Analysis for Identification of Mycobacteria Growing in Liquid Media  

Microsoft Academic Search

A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP

THERESA B. TAYLOR; CANDY PATTERSON; YVONNE HALE; ANDWILLIAM W. SAFRANEK

1997-01-01

286

Terminal-restriction fragment length polymorphism analysis of biphenyl dioxygenase genes from a polychlorinated biphenyl-polluted soil  

Microsoft Academic Search

Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that can be co-metabolically biotransformed by biphenyl-utilizing bacteria. In this study, terminal-restriction fragment length polymorphism (T-RFLP) was applied to the substrate specificity-determining region of the 2,3-biphenyl dioxygenase encoding genes of a microbial community found in a PCB-polluted soil. Notably, both the total biphenyl\\/PCB-utilizing community and its members actively expressing the 2,3-biphenyl dioxygenase

Serena Capodicasa; Stefano Fedi; Monica Carnevali; Leonardo Caporali; Carlo Viti; Fabio Fava; Davide Zannoni

2009-01-01

287

Genetic Heterogeneity in Mycobacterium tuberculosis Isolates Reflected in IS6110 Restriction Fragment Length Polymorphism Patterns as Low-Intensity Bands  

Microsoft Academic Search

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS6110 RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple M. tuberculosis strains. We did not find any multiple LIBs,

ANNETTE S. DE BOER; KRISTIN KREMER; MARTIEN W. BORGDORFF; PETRA E. W. DE HAAS; HERRE F. HEERSMA; DICK VAN SOOLINGEN

2000-01-01

288

Species-Specific Identification of Mycobacterium leprae by PCR-Restriction Fragment Length Polymorphism Analysis of the hsp65 Gene  

Microsoft Academic Search

PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene present in all mycobacteria was used in the present investigation to characterize Mycobacterium leprae. Bacilli were extracted and purified from different organs from experimentally infected armadillos and nude mice (Swiss mice of nu\\/nu origin). A total of 15 samples were assayed in duplicate, and the results were compared with those

NALIN RASTOGI; KHYE SENG GOH; MYLENE BERCHEL

1999-01-01

289

Analysis of DNA fragmentation using a dynamic size-sieving polymer solution in capillary electrophoresis.  

PubMed

Various natural and induced processes cause DNA fragmentation. Examples of these processes include apoptosis, enzymatic digestion, free radical production from ionizing radiation, photoscission by laser radiation and thermal degradation. Slab gel electrophoresis has been used most often to monitor such DNA damage. We have investigated with capillary electrophoresis the use of a new size-sieving polymer solution, TreviSol-CE (TS-CE), to monitor the DNA fragments produced from a variety of degradation processes. This polymer solution provides high run-to-run migration time and peak width reproducibilities and high separation efficiency of double-stranded DNA fragments in the 500 to 7000 base pair size range. Analysis of apoptotic DNA fragments suggested the presence of multiple nucleosomes within each cell type investigated. For irradiated DNA standards, peak-width-at-half-height and peak area were used to monitor the progress of DNA fragmentation. For both apoptotic DNA and irradiated DNA standards, fine structural features of fragmentation were revealed. PMID:9210317

Siles, B A; Nackerdien, Z E; Collier, G B

1997-05-30

290

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution.  

PubMed

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs. PMID:16061932

Müller, Kristian M; Stebel, Sabine C; Knall, Susanne; Zipf, Gregor; Bernauer, Hubert S; Arndt, Katja M

2005-08-01

291

Restriction enzyme analysis of the germ line limited DNA of Ascaris suum  

Microsoft Academic Search

The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33258 — CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The

Gtinther E. Roth; Karl B. Moritz

1981-01-01

292

The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations  

Microsoft Academic Search

Summary Restriction endonucleases and agarose gel electrophoresis have been used to demonstrate extensive nucleotide sequence diversity in mitochondrial DNA (mtDNA) within and between conspecific populations of rodents and other mammals. Cleavage of mtDNA samples with a relatively small number of endonucleases provides information concerning the phylogenetic relatedness of individual organisms which cannot now be readily obtained by any other type

Robert A. Lansman; Rosemary O. Shade; John F. Shapira; John C. Avise

1981-01-01

293

The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.  

PubMed

Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory. PMID:12514084

Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

294

Interactions between carbon nanotubes and DNA polymerase and restriction endonucleases  

NASA Astrophysics Data System (ADS)

Effects of multi-walled carbon nanotubes (MWCNT) and single-walled carbon nanotubes (SWCNT) functionalized with and without carboxylic groups on polymerase chain reaction (PCR) and restriction digestion reaction were investigated. The results showed that CNT can reduce and even inhibit PCR and restriction digestion reaction, possibly due to the decrease of respective enzyme activity. The inhibition effect on double restriction digestion reaction and PCR was increased in the order of CNT-COOH > pristine CNT and SWCNT> MWCNT. This study demonstrated that CNT may significantly affect the efficiency of biochemical reactions through different action mechanisms, which is critical for understanding how nanomaterials impact biological systems.

Yi, Changqing; Fong, Chi-Chun; Chen, Weiwei; Qi, Suijian; Tzang, Chi-Hung; Lee, Shuit-Tong; Yang, Mengsu

2007-01-01

295

Characterization of Microbial Diversity by Determining Terminal Restriction Fragment Length Polymorphisms of Genes Encoding 16S rRNA  

Microsoft Academic Search

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5* end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction

WEN-TSO LIU; TERENCE L. MARSH; HANS CHENG; LARRY J. FORNEY

1997-01-01

296

Characterization of western European field isolates and vaccine strains of avian infectious laryngotracheitis virus by restriction fragment length polymorphism and sequence analysis.  

PubMed

Infectious laryngotracheitis is a dramatic disease of the upper respiratory tract in poultry caused by a herpesvirus. In this study we investigated the characteristics of western European field isolates of infectious laryngotracheitis virus (ILTV) to gain more information on their diversity. The examined 104 isolates, collected from acute outbreaks during the last 35 years, originated from eight different countries: Switzerland (48), Germany (21), Sweden (14), the United Kingdom (9), Italy (5), Belgium (4), Austria (2), and Norway (1). Two vaccines, a chicken embryo origin product and a tissue culture origin product, were included in the survey. Polymerase chain reaction (PCR) was performed to amplify a 2.1-kb DNA fragment of ILTV using primers generated for the thymidine kinase (TK) gene. After digestion of the resulting PCR products by restriction endonuclease HaeIII, restriction fragment length polymorphism analysis was carried out. PCR amplicons of three field isolates and both vaccine strains were selected for sequencing. Here 98 field isolates showed the same cleavage pattern and were identical to both vaccine strains (clone 1). They differed from five Swiss isolates with identical cleavage pattern (clone 2) and one Swedish isolate (clone 3). The present study demonstrated that at least three clones of ILTV have been circulating in western Europe during the last 35 years. The 104 isolates analyzed showed a high genetic similarity regarding the TK gene, and a large majority of the field isolates (98/104) were genetically related to the vaccine strains. PMID:18646457

Neff, C; Sudler, C; Hoop, R K

2008-06-01

297

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.  

PubMed

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires. PMID:19780841

Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

2009-08-28

298

Caspase-2 cleaves DNA fragmentation factor (DFF45)/inhibitor of caspase-activated DNase (ICAD).  

PubMed

To investigate the signal transduction pathway of caspase-2, cell permeable Tat-reverse-caspase-2 was constructed, characterized and utilized for biochemical and cellular studies. It could induce the cell death as early as 2h, and caspase-2-specific VDVADase activity but not other caspase activities including DEVDase and IETDase. Interestingly, nuclear DNA fragmentation occurred and consistently DNA fragmentation factor (DFF45)/Inhibitor of caspase-activated DNase (ICAD) was cleaved inside the cell as well as in vitro, suggesting a role of caspase-2 in nuclear DNA fragmentation. PMID:17945178

Dahal, Giri Raj; Karki, Pratap; Thapa, Arjun; Shahnawaz, Mohammad; Shin, Song Yub; Lee, Jung Sup; Cho, Byungyun; Park, Il-Seon

2007-09-19

299

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution  

Microsoft Academic Search

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone sub- sequently cleaved with piperidine.

Kristian M. Muller; Sabine C. Stebel; Susanne Knall; Gregor Zipf; Hubert S. Bernauer; Katja M. Arndt

2005-01-01

300

Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes  

SciTech Connect

An early event in death of interphase lymphocytes exposed in vivo or in vitro to low doses of gamma-irradiation is the degradation of DNA into nucleosome-sized fragments. Induction of fragmentation required RNA and protein synthesis because actinomycin D and cycloheximide, respectively, are able to inhibit DNA fragmentation in irradiated lymphocytes. Studies adding cycloheximide and actinomycin D at various times postirradiation suggest that once the metabolic process is initiated within an individual cell it proceeds to completion. The reversible RNA synthesis inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits DNA fragmentation in irradiated thymocytes. When this drug is removed after 6 hr, irradiated thymocytes proceed to fragment their DNA; this suggests that an inducing signal that is not simply mRNA persists within the irradiated cell for at least 6 hr after irradiation. In contrast to mitogen-activated T and B lymphoblasts, resting T and B cells show significant DNA fragmentation after exposure to 100 to 500 rad. At 2000 rad, all of the splenic subpopulations die rapidly via a different mechanism. By studying the mechanism of DNA fragmentation induced during the interphase death of lymphocytes, we hope to understand better the extreme sensitivity of resting lymphocytes to radiation and what may be the common final pathway of programmed cell death.

Sellins, K.S.; Cohen, J.J.

1987-11-15

301

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA.  

PubMed

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region. PMID:11055916

Jasalavich, C A; Ostrofsky, A; Jellison, J

2000-11-01

302

CpG Methylation of DNA Restricts Prereplication Complex Assembly in Xenopus Egg Extracts  

Microsoft Academic Search

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded

Kevin J. Harvey; John Newport

2003-01-01

303

Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV.  

PubMed Central

We have isolated hybrid baculoviruses of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV) capable of replicating in both BmN (not susceptible to AcNPV) and SF-21 (not susceptible to BmNPV) cells (A. Kondo and S. Maeda, J. Virol. 65:3625-3632, 1991). Repeated backcross infection of one of these recombinant isolates with AcNPV generated eh-AcNPV, a virus with restriction endonuclease patterns of genomic DNA nearly identical to those of AcNPV but capable of replicating in both BmN and SF-21 cells, i.e., host range expanded. Expanded host range viruses were also isolated following cotransfection of AcNPV DNA with eh-AcNPV DNA cleaved with either HindIII or PstI. Subsequent cotransfection of AcNPV DNA with plasmids from an eh-AcNPV DNA fragment library identified an 11-kbp HindIII fragment that could expand the host range of AcNPV. Subcloning and cotransfection analyses localized a 572-bp SacI-HindIII fragment within this 11-kbp fragment which could alone expand the host range of AcNPV. Mapping and nucleotide sequencing analysis revealed that this fragment was identical to the corresponding 572-bp fragment (BmScH) of BmNPV. Furthermore, this fragment originated from the coding region of the putative DNA helicase gene. Cotransfection of AcNPV DNA with BmScH also generated a host range-expanded virus, eh2-AcNPV. These results indicated that the expanded host range characteristics of eh2-AcNPV were solely the result of recombination within the coding region of the putative DNA helicase gene. Images

Maeda, S; Kamita, S G; Kondo, A

1993-01-01

304

Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping  

Microsoft Academic Search

Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these sys- tems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work sug- gested that the

Kelly J. Neaves; Laurie P. Cooper; John H. White; Stewart M. Carnally; David T. F. Dryden; J. Michael Edwardson; Robert M. Henderson

2009-01-01

305

Cleavage maps for human cytomegalovirus DNA strain AD169 for restriction endonucleases EcoRI, BglII, and HindIII.  

PubMed Central

We have used cloned EcoRI fragments of the human CMV (HCMV) genome, strain AD169, to prepare restriction endonuclease maps of the DNA. Individual 32P-labeled cloned fragments were hybridized to Southern blots of HCMV DNA cleaved to completion with the restriction endonucleases BglII and HindIII and cleaved partially with EcoRI. By determining which EcoRI fragments hybridized to the same band on a Southern blot, we were able to establish linkage groups. This information coupled with the data derived from digestion of the cloned fragments with the enzymes BglII and HindIII (Tamashiro et al., J. Virol. 42:547-557, 1982) provided the basis for the construction of detailed maps for the enzymes EcoRI, BglII, and HindIII. We also identified the EcoRI fragments derived from the termini of this genome and mapped them with respect to the BglII and HindIII terminal fragments. From our mapping data, we conclude that the genome of HCMV is approximately 240 kilobases in length and is divided into long (198 kilobases) and short (42 kilobases) regions. Both regions consist of a unique sequence bounded by inverted repeats (11 to 12 kilobases for the long region and 2 to 3 kilobases for the short region). Furthermore, the long and short regions can invert relative to each other. Images

Spector, D H; Hock, L; Tamashiro, J C

1982-01-01

306

A Semester-Long Project for Teaching Basic Techniques in Molecular Biology Such as Restriction Fragment Length Polymorphism Analysis to Undergraduate and Graduate Students  

PubMed Central

Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky73. Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers.

DiBartolomeis, Susan M.

2011-01-01

307

Polymerase chain reaction-restriction fragment length polymorphism of mitochondrial 12S rRNA gene: a simple method for identification of poultry meat species.  

PubMed

Chicken (Gallus gallus), duck (Anas platyrhynchos), turkey (Meleagris gallopavo), guinea fowl (Numida meleagris) and quail (Coturnix japonica) are the common poultry species consumed as meat throughout the world. In this work, a molecular technique has been developed for identification and differentiation of meat originating from these species. This tool helps in detection of misrepresentation of different poultry meats. The technique involves the extraction of DNA from the given sample, polymerase chain reaction (PCR) amplification of mitochondrial 12S rRNA gene using universal primers, restriction analysis with selected restriction enzymes, followed by identification of meat species based on restriction fragment length polymorphism (RFLP) pattern. In this study, we used HinfI, Mph1 103I, MvaI, and Eco47I to identify and differentiate to poultry species referred to above. This species identification technique has also been applied successfully to processed meat products including those cooked at 120 degrees C for 30 min. Simplicity of interpretation of results combined with versatility makes this a convenient and appropriate technique in the hands of meat analysts for identifying poultry meat species. PMID:17253115

Girish, P S; Anjaneyulu, A S R; Viswas, K N; Santhosh, F H; Bhilegaonkar, K N; Agarwal, R K; Kondaiah, N; Nagappa, K

2007-05-01

308

An Asymmetric Complex of Restriction Endonuclease MspI on Its Palindromic DNA Recognition Site  

Microsoft Academic Search

Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence. Unlike any other

Qian Steven Xu; Rebecca B. Kucera; Richard J. Roberts; Hwai-Chen Guo

2004-01-01

309

Heterothallic species of neurospora are distinguishable by restriction analysis of their nuclear rDNA sequences  

Microsoft Academic Search

Restriction analysis of rDNAs was used to distinguish nuclear rDNA's of three different reference strains of heterothallic species of the genus Neurospora: N. crassa 74A (FGSC number987), N. intermedia P420 (FGSC number2316), and N. sitophila 10B (FGSC number580). Two approaches were adopted: (1) Nuclear DNA's of these three Neurospora species were treated with various restriction enzymes. Against the streaks of

C. Chambers; S. K. Dutta

1983-01-01

310

Characterization of Orientia tsutsugamushi strains isolated in Shandong Province, China by immunofluorescence and restriction fragment length polymorphism (RFLP) analyses.  

PubMed

In order to identify the characteristics of the Sta56 gene of the 23 isolates of Orientia (O.) tsutsugamushi isolated in Shandong Province, indirect immunofluorescence assay (IFA) was used to identify the gene type of 23 strains O. tsutsugamushi isolated from scrub typhus patients, chigger mites, and rodents. Restriction fragment length polymorphism (RFLP) analysis was also used to analyze the restriction profiles of the Sta56 gene PCR amplification products of the 23 isolated strains of the O. tsutsugamushi; the results were compared with those acquired by nested PCR. By IFA, 21 of the 23 isolates belonged to the Gilliam type, and 2 to the Karp type. Using RFLP analysis, 21 strains had similar restriction profiles to the Japan Kawasaki strain, but they had no restriction site Hha I, and thus had some difference in gene sequence compared with the Japan Kawasaki strain. The other 2 strains had similar restriction profiles to Karp. These results were identical to that acquired by nested-PCR. In Shandong Province, the gene types of epidemic O. tsutsugamushi strains were similar to the Japan Kawasaki type, but had some differences in gene sequence. In addition, Karp also existed. PMID:15691135

Liu, Yun-xi; Zhao, Zhong-tang; Gao, Yuan; Jia, Chong-qi; Zhang, Jing-lan; Yang, Zhan-qing; Wang, Shu-mei; Jiang, Bao-fa

2004-06-01

311

Type III restriction enzymes need two inversely oriented recognition sites for DNA cleavage  

Microsoft Academic Search

TYPE III restriction\\/modification enzymes recognize short, non-palindromic sequences that can be methylated on only one strand, with the paradoxical consequence that during replication of what is in effect hemimethylated DNA totally unmodified sites arise1. Why the unmodified sites are not subject to suicidal restriction was not clear. Here we show that restriction requires two unmodified recognition sites that can be

Andreas Meisel; Thomas A. Bickle; Detlev H. Kriiger; Cornelia Schroeder

1992-01-01

312

Clusters of DNA damage induced by ionizing radiation: Formation of short DNA fragments. I. Theoretical modeling  

SciTech Connect

We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber composed of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and {delta} rays due to knock-on collisions involving energy transfers > 100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of {circ}OH, {circ}H, e{sub aq}, etc.; {circ}OH attack on sugar molecules leading to strand breaks; {circ}OH attack on bases; direct ionization of the sugar molecules leading to strand breaks; direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 hp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the chromatin fibers in mammalian DNA. 27 refs., 7 figs.

Holley, W.R.; Chatterjee, A. [Lawrence Berkeley National Laboratory, Berkeley, CA (United States)

1996-02-01

313

PHYLOGENETIC RELATIONSHIPS OF SUBTRIBE ECLIPTINAE (ASTERACEAE :H ELIANTHEAE) BASED ON CHLOROPLAST DNA RESTRICTION SITE DATA1  

Microsoft Academic Search

Phylogenetic analysis of chloroplast DNA restriction site data for 76 of the 302 genera of Heliantheae sensu lato using 16 restriction endonucleases reveals that subtribe Ecliptinae is polyphyletic and that its genera are distributed in four different lineages. The ecliptinous genera Squamopappus, Podachaenium, Verbesina , and Tetrachyron (of the Neurolaeninae), along with other members of subtribe Neurolaeninae are the basalmost

JOSE L. PANERO; ROBERT K. JANSEN; JENNIFER A. CLEVINGER

314

Definition of restriction, Paul BergSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Interviewee: Paul Berg DNAi Location:Manipulation>Techniques>Cutting & Pasting>On the phenomenon of restriction On the phenomenon of restriction Paul Berg speaks about Herbert Boyer's research into the process by which an organism, such as a bacterium, can recognize and destroy foreign DNA.

2008-03-26

315

Synthesis of phosphorothioate-containing DNA fragments by a modified hydroxybenzotriazole phosphotriester approach.  

PubMed Central

The phosphorothioylating agent which was obtained by treating 2,5-dichlorophenyl phosphorodichloridothioate with 1-hydroxy-6-nitrobenzotriazole proved to be very effective for the synthesis in solution and on a solid support of phosphorothioate-containing DNA fragments.

Marugg, J E; van den Bergh, C; Tromp, M; van der Marel, G A; van Zoest, W J; van Boom, J H

1984-01-01

316

Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes  

SciTech Connect

The authors molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passage N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-1/sup n/ and Fv-1/sup b/ cells indicated that the Fv-1 host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in pol gene (2.9 kilobase pairs from the left end of the map).

Boone, L.R.; Myer, F.E.; Yang, D.M.; Ou, C.Y.; Koh, C.K.; Roberson, L.E.; Tennant, R.W.; Yang, W.K.

1983-10-01

317

Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes  

SciTech Connect

Unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and invitro passaged N-tropic Gross (passage A) murine leukemia retroviruses were molecularly cloned. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-1/sup n/ and Fv-1/sup b/ cells indicated the the Fv-1 host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map.

Boone, L.R.; Myer, F.E.; Yang, D.M.; Ou, C.Y.; Koh, C.K.; Roberson, L.E.; Tennant, R.W.; Yang, W.K.

1983-10-01

318

Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay  

Microsoft Academic Search

Meta-analyses were conducted to investigate the relationship of sperm DNA fragmentation on pregnancy outcome using in-vivo fertilization, IUI, routine IVF and ICSI. Couples with no known infertility problems were 7.0 times (CI 3.17, 17.7) more likely to achieve a pregnancy\\/delivery if the DNA fragmentation index (DFI) was <30% (n = 362, P = 0.0001) using in-vivo fertilization. Infertile couples using

Donald Evenson; Regina Wixon

2006-01-01

319

Chromosomal localization and genomic organization of cloned repetitive DNA fragments in mosquitoes (Diptera: Culicidae)  

Microsoft Academic Search

The chromosomal localization and genomic organization of three cloned repetitive DNA fragments (viz., H-76, H-61, and H-19)\\u000a isolated from theAedes albopictus genome have been examined inAe. albopictus and six otherAedes species:Ae. aegypti, Ae. seatoi, Ae. flavopictus, Ae. polynesiensis, Ae. alcasidi andAe. katherinensis. The results fromin situ and Southern hybridization analyses show that the sequences homologous to cloned repetitive DNA fragments

A. Kumar; K. S. Rai

1991-01-01

320

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns  

Microsoft Academic Search

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine\\u000a population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six\\u000a enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the\\u000a Philippine population of native cattle, indicating the

Tomomasa Watanabe; Joseph S. Masangkay; Shigeharu Wakana; Naruya Saitou; Takeshi Tomita

1989-01-01

321

Investigations of Bacterial Inactivation and DNA Fragmentation Induced by Flowing Humid Argon Post-discharge  

NASA Astrophysics Data System (ADS)

Bio-contaminated surfaces were exposed to an atmospheric pressure flowing post-discharge, i.e. without direct contact of the plasma with the surface. The non-thermal plasma source was a dielectric barrier discharge. Using humid argon as a feed gas, a reduction of six orders of magnitude of survivors could be obtained for Escherichia coli. An investigation of bacterial inactivation mechanisms during the plasma induced treatment was conducted. For this purpose, DNA (plasmid and genomic DNA in aqueous solution) degradation by the plasma process was studied, assuming that the bacterial inactivation is obtained when the bacterial DNA is fragmented. According to the operating conditions (feed gas, reactor geometry and discharge input power), DNA fragmentation was evaluated in correlation with aqueous phase hydrogen peroxide concentration measurements. It appears that hydrogen peroxide is not the only factor responsible for DNA fragmentation and that short-lived species produced by water dissociation are major contributors.

Odic, Emmanuel; Limam, S.; Kirkpatrick, M. J.; Dodet, B.; Salamitou, S.; DuBow, M. S.

322

APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS  

PubMed Central

Objective: To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes. Materials and Methods: Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods. Results: PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%. Conclusion: PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes.

Bagyalakshmi, R; Senthilvelan, B; Therese, K L; Murugusundram, S; Madhavan, H N

2008-01-01

323

Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis.  

PubMed Central

The HpaI E fragment (0-4.5 map units) of adenovirus type 2 (Ad2) DNA was cloned in the plasmid vector pBR322. Excision of the viral insert with PstI and XbaI generated a fragment which comigrated with Ad2 XbaI-E (0-3.8 map units), and this fragment was ligated to the 3.8-100 fragment generated by XbaI cleavage of the DNA of the Ad5 mutant, dl309 (N. Jones and T. Shenk, Cell 17:683-689, 1979). Transfection with the ligation products resulted in the production of progeny virus which was able to replicate on both HeLa and line 293 cells, demonstrating the biological activity of the sequences rescued from the plasmid. Small deletions were introduced around the SmaI site (map position 2.8) within the cloned viral insert, and the altered DNA sequences were reintroduced into progeny virus as described above. The mutant viruses grew well on line 293 cells but plaqued with greatly reduced efficiency on HeLa cells, exhibiting a host range phenotype similar to previously described mutants with lesions located within this region of the genome. When plasmid-derived left-end fragments containing pBR322 DNA sequences to the left of map position 0 were ligated to the 3.8-100 fragment of dl309 DNA, the infectivity of the ligation products was not reduced. However, all progeny viruses examined yielded normal-size restriction enzyme fragments from their left-hand ends, indicating that the bulk of the pBR322 DNA sequences are removed either prior to or as a consequence of the replication of the transfecting DNA molecules. Images

Stow, N D

1981-01-01

324

RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF PCR-AMPLIFIED NIFH SEQUENCES FROM WETLAND PLANT RHIZOSPHERE COMMUNITIES  

EPA Science Inventory

We describe a method to assess the community structure of N2-fixing bacteria in the rhizosphere. Total DNA was extracted from Spartina alterniflora and Sesbania macrocarpa root zones by bead-beating and purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 ...

325

Restriction and gene maps of plastid DNA from Capsicum annuum  

Microsoft Academic Search

Chloroplasts and chromoplasts were isolated from green and red fruits, respectively, of the bell pepper, Capsicum annuum var. Emerald giant. A comparison of the restriction patterns of DNAs isolated from these plastids was made using single and double digests by SacI, PvuII, PstI, and SalI and found to be indistinguishable. It is inferred therefore that the conversion of chloroplasts to

Ioannis Gounaris; Christine B. Michalowski; Hans J. Bohnert; Carl A. Price

1986-01-01

326

Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.  

PubMed

Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART. PMID:23230887

Balasuriya, A; Serhal, P; Doshi, A; Harper, J C

2012-12-11

327

Genetic characterization of E2 gene of classical swine fever virus by restriction fragment length polymorphism and phylogenetic analysis  

Microsoft Academic Search

An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for\\u000a genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified\\u000a 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003–2008. The PCR products of positive\\u000a samples were further differentiated using MspI digestion, 23 were identified

Ning Chen; Dejiang Li; Xuemei Yuan; Xiaoliang Li; Hongxia Hu; Binglin Zhu; Xiaoyuan Wan; Weihuan Fang

2010-01-01

328

Kinetic Models of Translocation, Head-On Collision, and DNA Cleavage by Type I Restriction Endonucleases †  

Microsoft Academic Search

Digestion of linear DNA by type I restriction endonucleases is generally activated following the head-on collision of two translocating enzymes. However, the resulting distributions of cleavage loci along the DNA vary with different enzymes; in some cases, cleavage is located in a discrete region midway between a pair of recognition sites while in other cases cleavage is broadly distributed and

Mark D. Szczelkun

2002-01-01

329

A homology model of restriction endonuclease SfiI in complex with DNA  

Microsoft Academic Search

BACKGROUND: Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very

Agnieszka A Chmiel; Janusz M Bujnicki; Krzysztof J Skowronek

2005-01-01

330

Restriction endonuclease DNA fingerprinting of respiratory, foetal and perinatal foal isolates of equine herpesvirus type 1  

Microsoft Academic Search

Summary DNA was prepared from 43 equine herpesvirus type 1 (EHV1) isolates, 11 of which were from horses with respiratory disease, 22 from aborted equine foetuses, and 10 from foals that died perinatally. The restriction endonuclease DNA fingerprints of 10 of the 11 respiratory isolates, known with certainty to have been recovered from horses with respiratory disease, were entirely different

M. J. Studdert

1983-01-01

331

Characterization of Mitochondrial DNA in Various Candida Species: Isolation, Restriction Endonuclease Analysis, Size, and Base Composition  

Microsoft Academic Search

A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and

CHYONG-SHWU SU; SALLY A. MEYER

1991-01-01

332

Effect of restriction endonucleases on assessment of biodiversity of cultivable polar marine planktonic bacteria by amplified ribosomal DNA restriction analysis.  

PubMed

To choose a suitable restriction endonuclease for quick assessment of bacterial diversity in polar environments by ARDRA, we investigated the effect of restriction enzymes on ARDRA patterns of cultivable marine planktonic bacteria isolated from polar region. Thirty-three isolates were analyzed by ARDRA using five enzymes (HinfI, HaeIII, AluI, and the mix AfaI/MspI), respectively, resulting in different groups, each group corresponding to a particular genotype. A comparison of the ARDRA patterns was carried out, and phylogenetic position of all thirty-three bacteria was obtained by 16S rDNA sequencing. Consistent with phylogenetic analysis, ARDRA pattern comparison revealed that AluI, being sensitive and reliable enough to generate species-specific patterns, was a suitable restriction enzyme used for evaluating bacterial diversity, suggesting a combination of ARDRA with AluI and 16S rDNA sequencing can provide a simple, fast and reliable means for bacterial identification and diversity assessment in polar environments. PMID:17502990

Zeng, Yinxin; Liu, Wenqi; Li, Huirong; Yu, Yong; Chen, Bo

2007-05-15

333

Effect of dietary restriction upon the age-associated decline of lymphocyte DNA repair activity in mice  

Microsoft Academic Search

Effects of dietary restriction (DR) on DNA repair capacity of mouse splenocytes after ultraviolet (UV)-induced damage were\\u000a assessed. Two mouse cohorts received restricted amounts of purified hypocaloric diets; one was minimally restricted (?75%\\u000a of the caloric intake of mice fed a commercial diet ad libitum), the other was severely restricted (?50% caloric restriction). An inverse correlation between age and DNA

Federico Licastro; Richard Weindruch; L. Jane Davis; Roy L. Walford

1988-01-01

334

Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis  

PubMed Central

The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662–1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

Dunbar, John; Ticknor, Lawrence O.; Kuske, Cheryl R.

2000-01-01

335

Use of mgc2-polymerase chain reaction-restriction fragment length polymorphism for rapid differentiation between field isolates and vaccine strains of Mycoplasma gallisepticum in Israel.  

PubMed

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment length polymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries. PMID:16094829

Lysnyansky, Inna; Garcia, Maricarmen; Levisohn, Sharon

2005-06-01

336

DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction  

Microsoft Academic Search

DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but

Ann-Marie Bröske; Lena Vockentanz; Shabnam Kharazi; Matthew R Huska; Elena Mancini; Marina Scheller; Christiane Kuhl; Andreas Enns; Marco Prinz; Rudolf Jaenisch; Claus Nerlov; Achim Leutz; Miguel A Andrade-Navarro; Sten Eirik W Jacobsen; Frank Rosenbauer

2009-01-01

337

Restriction endonuclease cleavage site map of safflower ( Carthamus tinctorius L.) chloroplast DNA  

Microsoft Academic Search

The restriction endonucleases SalI, PstI, KpnI and HindIII have been used to construct a physical map of safflower (Carthamus tinctorius L.) chloroplast DNA. This was accomplished by hybridizing Southern blots of single and double digested chloroplast DNA with 32P-dCTP nick-translated SalI, KpnI and HindIII probes which were individually isolated from agarose gels. The chloroplast DNA was found to be circular

M. A. Smith; C. Ma

1985-01-01

338

Unique P Spectral Response to the Formation of a Specific Restriction Enzyme–DNA Complex  

Microsoft Academic Search

Protein-induced distortion is a dramatic but not universally observed feature of sequence-specific DNA interactions. This is illustrated by the crystal structures of restriction enzyme–DNA complexes: While some of these structures exhibit DNA distortion, others do not. Among the latter is PvuII endonuclease, a small enzyme that is also amenable to NMR spectroscopic studies. Here P NMR spectroscopy is applied to

Cynthia M. Dupureur

2006-01-01

339

Structure, restriction map and infectivity of the genomic and replicative forms of Aa PV DNA  

Microsoft Academic Search

Summary We have characterized the genomic and replicative form (RF) DNA of theAedes albopictus Parvovirus (AaPV), a virus isolated from a chronically infected C6\\/36 clone ofAedes albopictus cell line [22]. The genome ofAaPV virions is a single-stranded linear DNA molecule approximately 4.2 kb in length, essentially (about 90%) encapsidated as minus strand. A restriction map of the RF DNA isolated

Y. Boublik; F.-X. Jousset; M. Bergoin

1994-01-01

340

Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation  

Microsoft Academic Search

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Cur- rently, its existence is inferred mainly from gel elec- trophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the devel- opment of a

Yael Gavrieli; Yoav Sherman; Shmuel A. Ben-Sasson

1992-01-01

341

Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.  

PubMed Central

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

Jin, H; Ren, Z; Pozsgay, J M; Elkins, C; Whitby, P W; Morton, D J; Stull, T L

1996-01-01

342

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil).  

PubMed

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC6 indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene. PMID:16738861

Yamada, Tetsuya; Takatsu, Yasumasa; Kasumi, Masakazu; Ichimura, Kazuo; van Doorn, Wouter G

2006-11-01

343

Effects of 2-chloroadenine substitution in DNA on restriction endonuclease cleavage reactions.  

PubMed

The purine analog, 2-chloro-2'-deoxyadenosine triphosphate (CldATP), was incorporated enzymatically in place of dATP into the minus strand of M13mp18 duplex DNA. Its effect on protein-DNA interactions was assessed by determining the amount of DNA cleavage by type II restriction endonucleases. Substitution of chloroadenine (CIAde) for adenine (Ade) in DNA appreciably decreased the amount and rate of DNA cleavage of the minus strand when the analog was situated within the appropriate endonuclease recognition site. CIAde residues flanking a restriction site had variable effects. SmaI cleaved both CIAde-containing and control substrates with equal efficiency. NarI, however, was stimulated 1.5-fold by the presence of CIAde outside its recognition site. The effects of analog incorporation on restriction enzyme cleavage of an opposing unsubstituted strand of duplex DNA was examined by enzymatically incorporating CIdATP into the complementary minus strand of a 36-base oligonucleotide. Endonucleolytic cleavage of both plus and minus strands was reduced on 36-mers containing CIAde residues located within only the minus strand. These data suggest that CIAde residues incorporated into a single DNA strand may have an appreciable effect on DNA-protein interactions that involve one or both strands of duplex DNA. PMID:1647525

Hentosh, P; McCastlain, J C

1991-06-11

344

Effects of ulipristal acetate on sperm DNA fragmentation during in vitro incubation.  

PubMed

Abstract Objective Ulipristal acetate (UPA) acts as an emergency contraceptive by inhibiting ovulation. This study explores possible additional effects on the fragmentation of sperm DNA during in vitro incubation. Methods Motile spermatozoa from healthy donors were selected by swim-up and incubated under capacitating conditions in control medium or with UPA (1, 10, 100, 1,000 or 10,000 ng/ml). In some experiments, 200 ?M of H2O2 were added to induce oxidative stress. The sperm chromatin dispersion test was performed to analyse DNA integrity (400 cells; 1000×). Lipid peroxidation (thiobarbituric acid assay), induced-acrosome reaction (AR) and sperm vitality (Eosin Y) were also evaluated in spermatozoa exposed to UPA and/or H2O2. Results During sperm incubation, the percentage of fragmented DNA increased significantly, from 15.0 ± 1.3 to 41.0 ± 4.5% (p < 0.001). In the presence of UPA, DNA fragmentation decreased significantly (p < 0.05), in a dose-dependent manner. At 100 and 1000 ng/ml, UPA also counteracted the effect of H2O2 and prevented DNA fragmentation. No effect on sperm vitality, lipid peroxidation or induced-AR was found with any treatment. Conclusions During in vitro sperm capacitation DNA fragmentation increased but the latter was counteracted in the presence of UPA, which possibly acted as a scavenger of reactive oxygen species produced by spermatozoa. PMID:23879383

Munuce, María José; Cicaré, Juliana; Zumoffen, Carlos; Caille, Adriana; Ghersevich, Sergio; Bahamondes, Luis

2013-07-24

345

Crystallographic Studies of Protein-Nucleic Acid Interaction: Catabolite Gene Activator Protein and the Large Fragment of DNA Polymerase I  

Microsoft Academic Search

Crystals suitable for X-ray crystallographic investigation have been grown of several nucleic acid binding proteins and their analysis is in progress. These include E. coli catabolite gene activator protein (CAP), the large fragment of DNA polymerase I (Pol I fragment), rec A, single strand DNA binding protein, resolvase, lac repressor and lac repressor ‘Core’, 5S RNA fragment and its complex

T. A. Steitz; I. T. Weber; D. Ollis; P. Brick

1983-01-01

346

A simple method for isolation of DNA fragments associated with the nuclear lamina in vivo.  

PubMed

We describe a simple method for the purification of DNA fragments associated with the nuclear lamina in vivo. Ehrlich ascite tumor cells are first u.v.-irradiated to crosslink DNA to proteins. The nuclear lamina is then isolated and purified by low-speed centrifugation through a cushion of 40% sucrose. The material sedimenting through the created density barrier represents nuclear lamina of a very high purity, free from any DNA fragments except those which were in a crosslinking distance to it in vivo. PMID:2313033

Christova, R; Galcheva-Gargova, Z

1990-01-01

347

Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.  

PubMed Central

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity. Images

Moyer, C L; Dobbs, F C; Karl, D M

1994-01-01

348

Cyclization of short DNA fragments and bending fluctuations of the double helix  

PubMed Central

Cloutier and Widom [Cloutier, T. E. & Widom, J. (2004) Mol. Cell 14, 355–362] recently reported that the cyclization efficiency of short DNA fragments, about 100 bp in length, exceeds theoretical expectations by three orders of magnitude. In an effort to resolve this discrepancy, we tried modifying the theory. We investigated how the distribution of the angles between adjacent base pairs of the double helix affects the cyclization efficiency. We found that only the incorporation of sharp kinks in the angle distribution provides the desired increase of the cyclization efficiency. We did not find a model, however, that fits all cyclization data for DNA fragments of different lengths. Therefore, we carefully reinvestigated the cyclization of 100-bp DNA fragments experimentally and found their cyclization efficiency to be in remarkable agreement with the traditional model of DNA bending. We also found an explanation for the discrepancy between our results and those of Cloutier and Widom.

Du, Quan; Smith, Chaim; Shiffeldrim, Nahum; Vologodskaia, Maria; Vologodskii, Alexander

2005-01-01

349

Human immunoglobulin heavy chain A2 gene allotype determination by restriction fragment length polymorphism.  

PubMed Central

The human immunoglobulin heavy chain alpha 2 genes have two allelic forms or allotypes called A2m(1) and A2m(2). Previously, these allotypic markers have only been distinguishable by serology. Studies of the alpha 2 genes, however, show that it is possible to differentiate between the allotypes by restriction enzyme site polymorphisms, both in the protein coding regions and in flanking regions. These polymorphic sites have been used to determine the alpha 2 allotypes of several human DNAs. Images

Lefranc, M P; Rabbitts, T H

1984-01-01

350

Simulation of DNA fragment distributions after irradiation with photons  

Microsoft Academic Search

The Monte Carlo track structure code PARTRAC has been further improved by implementing electron scattering cross-sections\\u000a for liquid water and by explicitly modelling the interaction of water radicals with DNA. The model of the genome inside a\\u000a human cell nucleus in its interphase is based on the atomic coordinates of the DNA double helix with an additional volume\\u000a for the

W. Friedland; Peter Jacob; Herwig G. Paretzke; Matteo Merzagora; Andrea Ottolenghi

1999-01-01

351

Multimerization-cyclization of DNA fragments as a method of conformational analysis.  

PubMed Central

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.

Podtelezhnikov, A A; Mao, C; Seeman, N C; Vologodskii, A

2000-01-01

352

Diagnosis and Species Identification of Mycobacterial Infections by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of Sterile Body Fluids  

PubMed Central

Background/Aims The development of effective, accurate, and rapid diagnostic methods for Mycobacterium infection and mycobacterial species identification is required. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is an easy, rapid and inexpensive technique for identifying Mycobacterium spp. Methods We performed PCR-RFLP to detect and identify Mycobacterium spp. from 10 sterile body fluids, including ascites, cerebrospinal fluid, pleural fluid, synovial fluid, and peritoneal dialysis fluid. Clinical samples were collected from patients with diagnoses of definite, probable or suspected mycobacterial infection. The conserved RNA polymerase genes of Mycobacterium spp. were amplified by PCR. Results The amplified 360-bp region of rpoB was digested with the restriction enzyme MspI or HaeIII. The PCR-RFLP results for the clinical samples were identical to those for M. tuberculosis, M. fortuitum, M. intracellulare, and M. avium. In addition, the results of the PCR-RFLP were identical to those obtained by DNA sequencing. Conclusions PCR-RFLP analysis of sterile body fluids may be a useful method for the diagnosis of mycobacterial infections and for the differentiation of mycobacterial species.

Cho, Cheong Ho; Han, Sang Hoon; Chin, Bum Sik; Choi, Suk Hoon; Lee, Han Sung; Kim, Chang Oh; Kim, Myung Soo; Choi, Jun Yong; Song, Young Goo

2009-01-01

353

Who is the mother of the potato? — restriction endonuclease analysis of chloroplast DNA of cultivated potatoes  

Microsoft Academic Search

Chloroplast DNA from 44 lines of 16 wild and 7 cultivatedSolanum species were compared by restriction endonuclease analysis. Seven chloroplast genome types were identified among them by 5 restriction enzymes: Type A (S. tuberosum ssp.andigena andS. maglia); Type S (S. goniocalyx, S. phureja, S. stenotomum, S. ×chaucha and a line of ssp.andigena); Type C (S. acaule, S. bukasovii, S. canasense,

K. Hosaka

1986-01-01

354

Tagging Developmental Genes in Dictyostelium by Restriction Enzyme-Mediated Integration of Plasmid DNA  

Microsoft Academic Search

Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid

Adam Kuspa; William F. Loomis

1992-01-01

355

Ionizing radiation-induced fragmentation of plasmid DNA Atomic force microscopy and biophysical modeling  

NASA Astrophysics Data System (ADS)

It is widely accepted that DNA double-strand breaks (DSBs) are closely correlated with radiation-induced cell killing and are the most critical lesions related to cellular endpoints like mutagenesis and transformation. High linear energy transfer (LET) radiation produces more severe and complex damage due to the fact that induced DSBs are not randomly distributed but clustered at different levels of DNA organization. In this paper, direct visualization of DSBs induced in a plasmid supercoiled DNA by low- and high-LET radiation is presented. Resulting DNA fragments distributions obtained by use of atomic force microscopy (AFM) are shown. Moreover, a biophysical model of spatially correlated DSBs formation in the framework of the Local Effect Model (LEM) is introduced and its predictions on DNA fragment formation are discussed.

Psonka, K.; Gudowska-Nowak, E.; Brons, S.; Elsässer, Th.; Heiss, M.; Taucher-Scholz, G.

356

Restriction fragment length polymorphism of mitochondrial genome of the entomopathogenic fungus Beauveria bassiana reveals high intraspecific variation.  

PubMed

Beauveria bassiana is an entomopathogenic fungus with a growing potential for pest control in different agro-ecosystems worldwide. Such potential brings the necessity of developing a strain specific typing system. In a previous study, we reported the identification of molecular variants in mitochondrial DNA (mtDNA) polymorphism in 15 North American isolates. Results indicated a highly conserved mitochondrial genome showing only two mitochondrial genotypes (mitotypes). In this study we used whole genomic DNA from 18 isolates of B. bassiana, two unidentified Beauveria spp., and one each of B. amorpha, B. cylindrospora and B. nivea from more diverse origins. By doing single- and double-restriction enzyme digestion of total genomic DNA with EcoRI, and HindIII and then probing with BbmtE2, the predominance of mitotypes A and B was observed again, along with three newly described mitotypes (C to E). Additionally, by using whole B. bassiana mtDNA digested with HpaII as probe, we further demonstrate up to nine different mitotypes within B. bassiana. With either of the two probes, distinguished between members of the genus Beauveria and from Paecilomyces farinosus and Metarhizium anisopliae. Phylogenetic analysis could not however distinguish B. amorpha and B. nivea isolates from B. bassiana, suggesting a close genetic relation between the three species of the genus. Altogether, these results show high variability in mitochondrial genome, which can be useful as a reliable tool for the biopesticide industry for both species and isolate specific identification. PMID:15506018

Uribe, Daniel; Khachatourians, George G

2004-09-01

357

DNA fragmentation in developing lung fibroblasts exposed to Stachybotrys chartarum (atra) toxins.  

PubMed

Stachybotrys chartarum (atra) is a toxic mold that grows on water-damaged cellulose-based materials. Research has revealed also that inhalation of S. chartarum spores caused marked changes in respiratory epithelium, especially to developing lungs. We analyzed the epigenetic potential of S. chartarum spore toxins on developing rat lung fibroblasts using single cell gel electrophoresis (comet assay). Isolated fetal lung fibroblasts were exposed to S. chartarum spore toxins for 15 min, 3, 14, or 24 hr and control cells were exposed to saline under the same conditions. Cells were embedded in agarose, electrophoresed under alkaline conditions and silver stained. DNA damage was assessed in terms of fragmentation as measured by comet tail length (DNA migration) and intensity (% DNA contained within head and tail). Upon visual inspection, control fibroblasts showed no DNA fragmentation whereas S. chartarum-treated cells had definable comets of various degrees depending upon the time-course. Analyses of the comets revealed that exposure to S. chartarum spore toxins for at least 15 min to 14 hr, induced increased DNA fragmentation in a time-dependent manner. The fact that exposure to toxins for 24 hr showed less damage suggested that developing lung fibroblasts may have the capability of repairing DNA fragmentation. PMID:17534970

McCrae, K C; Rand, T G; Shaw, R A; Mantsch, H H; Sowa, M G; Thliveris, J A; Scott, J E

2007-07-01

358

Mitochondrial fission controls DNA fragmentation by regulating endonuclease G  

Microsoft Academic Search

Mitochondria constantly undergo fusion and fission that are necessary for the maintenance of organelle fidelity. However, growing evidence has shown that abnormal mitochondrial fusion and fission participate in the regulation of apoptosis. Mitochondrial fusion is able to inhibit apoptosis, whereas mitochondrial fission is involved in the initiation of apoptosis. It remains elusive as to whether mitochondrial fission can regulate DNA

Jincheng Li; Jing Zhou; Yanrui Li; Danian Qin; Peifeng Li

2010-01-01

359

Comprehensive Study On The Metastable Negative Ion Fragmentation Of Individual Dna Components And Larger Oligonucleotides  

NASA Astrophysics Data System (ADS)

Here we present a systematic study on the unimolecular decay pathways of the deprotonated building blocks of DNA and RNA to address the following questions: 1. Are the negative ion fragmentation patterns observed in the metastable decay of individual DNA components still evident when these are combined to larger oligonucleotides? 2. What is the significance of the charge location in determining the fragmentation pathways in the metastable decay process? 3. Are those metastable decay channels relevant in dissociative electron attachment to DNA components? To address these questions we have studied the fragmentation patterns of the deprotonated ribose and ribose 5'-monophosphate, the fragmentation patterns of the individual bases, all nucleosides and all 2'-deoxynucleosides as well as the individual nucleotides and several combinations of hexameric oligonucleotides. Furthermore, to understand the significance of the charge location in determining the fragmentation path in the metastable decay process of these deprotonated ions we have also studied modified uridine and guanosine. These have been modified to block different deprotonation sites and thus to control the initial step in the in the fragmentation process i.e. the site of deprotonation. In addition to our experimental approach we have also simulated the metastable fragmentation of the deprotonated uridine and 2'-deoxyguanosine to clarify the mechanisms and fragmentation patterns observed. Where data is available, the results are compared to dissociative electron attachment to DNA components and discussed in context to the underlying mechanism. Experiments on modified nucleosides where selected deprotonation sites have been blocked are used to verify the predicted reaction paths and imulations on uridine and 2'-deoxyguanosine are compared to the experimental results and used to shed light on the mechanisms involved.

Ingolfsson, O.; Flosadottir, H. D.; Omarsson, B.; Ilko, B.

2010-07-01

360

Fragmentation of DNA in a sub-microliter microfluidic sonication device.  

PubMed

Fragmentation of DNA is an essential step for many biological applications including the preparation of next-generation sequencing (NGS) libraries. As sequencing technologies push the limits towards single cell and single molecule resolution, it is of great interest to reduce the scale of this upstream fragmentation step. Here we describe a miniaturized DNA shearing device capable of processing sub-microliter samples based on acoustic shearing within a microfluidic chip. A strong acoustic field was generated by a Langevin-type piezo transducer and coupled into the microfluidic channel via the flexural lamb wave mode. Purified genomic DNA, as well as covalently cross-linked chromatin were sheared into various fragment sizes ranging from ?180 bp to 4 kb. With the use of standard PDMS soft lithography, our approach should facilitate the integration of additional microfluidic modules and ultimately allow miniaturized NGS workflows. PMID:23014736

Tseng, Qingzong; Lomonosov, Alexey M; Furlong, Eileen E M; Merten, Christoph A

2012-11-21

361

Performance of heuristic methods driven by chaotic dynamics for ATSP and applications to DNA fragment assembly  

NASA Astrophysics Data System (ADS)

Chaotic dynamics has been shown to be effective in improving the performance of combinatorial optimization algorithms. In this paper, the performance of chaotic dynamics in the asymmetric traveling salesman problem (ATSP) is investigated by introducing three types of heuristic solution update methods. Numerical simulation has been carried out to compare its performance with simulated annealing and tabu search; thus, the effectiveness of the approach using chaotic dynamics for driving heuristic methods has been shown. The chaotic method is also evaluated in the case of a combinatorial optimization problem in the real world, which can be solved by the same heuristic operation as that for the ATSP. We apply the chaotic method to the DNA fragment assembly problem, which involves building a DNA sequence from several hundred fragments obtained by the genome sequencer. Our simulation results show that the proposed algorithm using chaotic dynamics in a block shift operation exhibits the best performance for the DNA fragment assembly problem.

Kato, Tomohiro; Hasegawa, Mikio

362

BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium  

Microsoft Academic Search

Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium.

J. Song; J. M. Bradeen; S. K. Naess; J. P. Helgeson; J. Jiang

2003-01-01

363

Specific sperm defects are differentially correlated with DNA fragmentation in both normozoospermic and teratozoospermic subjects.  

PubMed

A positive effect of selecting spermatozoa under high magnification during intracytoplasmic sperm injection (ICSI) has been described, but a clear explanation has not been given yet. Previous works have shown that high magnification selected spermatozoa have significantly better chromatin status than unselected cells; on the other hand, it has been reported that spermatozoa with no morphological defects can also be negatively associated with embryo quality and pregnancy outcome attributable to DNA fragmentation. The aim of this study was to investigate whether sperm morphology is correlated with DNA fragmentation, both in normozoospermic and teratozoospermic patients. A prospective cohort study involving 32 subjects was recruited over a 3-month period. Spermatozoa were fixed on a slide for TUNEL assay and evaluated using an epifluorescent light microscope equipped with a video monitor. Single TUNEL-positive or -negative cells were evaluated for morphology at ×4400 magnification. Each spermatozoon was then classified according to morphological normalcy or specific defects. The median percentage of typical forms was 11 and 0%, in the normozoospermic and teratozoospermic groups respectively (p = 0.001). In normozoospermic samples, the percentage of TUNEL-positive morphologically normal spermatozoa was 4%. By comparison, spermatozoa showing a vacuolated head or a small non-oval head had a significantly higher incidence of DNA fragmentation in both groups (12 and 13%, 19 and 13% respectively; p < 0.05). In contrast, spermatozoa showing a pyriform head had a DNA fragmentation rate similar to typical forms (3 and 5%, in normozoospermic and teratozoospermic respectively). This study shows that specific defects evaluated in fixed spermatozoa under high-power magnification are more likely to be associated with DNA fragmentation. High-magnification evaluation of spermatozoa can therefore reduce the probability of selecting cells carrying fragmented DNA during ICSI. PMID:24115574

Mangiarini, A; Paffoni, A; Restelli, L; Ferrari, S; Guarneri, C; Ragni, G; Somigliana, E

2013-09-30

364

FragIdent - Automatic identification and characterisation of cDNA-fragments  

PubMed Central

Background Many genetic studies and functional assays are based on cDNA fragments. After the generation of cDNA fragments from an mRNA sample, their content is at first unknown and must be assigned by sequencing reactions or hybridisation experiments. Even in characterised libraries, a considerable number of clones are wrongly annotated. Furthermore, mix-ups can happen in the laboratory. It is therefore essential to the relevance of experimental results to confirm or determine the identity of the employed cDNA fragments. However, the manual approach for the characterisation of these fragments using BLAST web interfaces is not suited for larger number of sequences and so far, no user-friendly software is publicly available. Results Here we present the development of FragIdent, an application for the automatic identification of open reading frames (ORFs) within cDNA-fragments. The software performs BLAST analyses to identify the genes represented by the sequences and suggests primers to complete the sequencing of the whole insert. Gene-specific information as well as the protein domains encoded by the cDNA fragment are retrieved from Internet-based databases and included in the output. The application features an intuitive graphical interface and is designed for researchers without any bioinformatics skills. It is suited for projects comprising up to several hundred different clones. Conclusion We used FragIdent to identify 84 cDNA clones from a yeast two-hybrid experiment. Furthermore, we identified 131 protein domains within our analysed clones. The source code is freely available from our homepage at .

Seelow, Dominik; Goehler, Heike; Hoffmann, Katrin

2009-01-01

365

Host factor SAMHD1 restricts DNA viruses in non-dividing myeloid cells.  

PubMed

SAMHD1 is a newly identified anti-HIV host factor that has a dNTP triphosphohydrolase activity and depletes intracellular dNTP pools in non-dividing myeloid cells. Since DNA viruses utilize cellular dNTPs, we investigated whether SAMHD1 limits the replication of DNA viruses in non-dividing myeloid target cells. Indeed, two double stranded DNA viruses, vaccinia and herpes simplex virus type 1, are subject to SAMHD1 restriction in non-dividing target cells in a dNTP dependent manner. Using a thymidine kinase deficient strain of vaccinia virus, we demonstrate a greater restriction of viral replication in non-dividing cells expressing SAMHD1. Therefore, this study suggests that SAMHD1 is a potential innate anti-viral player that suppresses the replication of a wide range of DNA viruses, as well as retroviruses, which infect non-dividing myeloid cells. PMID:23825958

Hollenbaugh, Joseph A; Gee, Peter; Baker, Jonathon; Daly, Michele B; Amie, Sarah M; Tate, Jessica; Kasai, Natsumi; Kanemura, Yuka; Kim, Dong-Hyun; Ward, Brian M; Koyanagi, Yoshio; Kim, Baek

2013-06-27

366

Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization  

PubMed Central

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.

Young, Douglas D.; Lusic, Hrvoje; Lively, Mark O.; Deiters, Alexander

2009-01-01

367

Development of a multiplexed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify common members of the Subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala.  

PubMed

Morphological differentiation of mosquitoes in the subgenera Culex (Culex) and Culex (Phenacomyia) in Guatemala is difficult, with reliable identification ensured only through examination of larval skins from individually reared specimens and associated male genitalia. We developed a multiplexed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify common Cx. (Cux.) and Cx. (Phc.). Culex (Cux.) chidesteri, Cx. (Cux.) coronator, Cx. (Cux.) interrogator, Cx. (Cux.) quinquefasciatus, Cx. (Cux.) nigripalpus/Cx. (Cux.) thriambus, and Cx. (Phc.) lactator were identified directly with a multiplexed primer cocktail comprising a conserved forward primer and specific reverse primers targeting ribosomal DNA (rDNA). Culex nigripalpus and Cx. thriambus were differentiated by restriction digest of homologous amplicons. The assay was developed and optimized using well-characterized specimens from Guatemala and the United States and field tested with unknown material from Guatemala. This assay will be a valuable tool for mosquito identification in entomological and arbovirus ecology studies in Guatemala. PMID:20682869

Kent, Rebekah J; Deus, Stephen; Williams, Martin; Savage, Harry M

2010-08-01

368

A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudo medicinalis and its expression in E. coli.  

PubMed Central

A DNA containing the coding sequence for the proteinase inhibitor protein, eglin c, from the leech Hirudo medicinalis has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 232 base-pair fragment containing initiation and termination codon signals with restriction enzyme recognition sites conveniently placed for cloning into a plasmid vector. Only six oligonucleotides from 34 to 61 bases in length, sharing pairwise stretches of complementary regions at their 3'-termini, were prepared by phosphotriester solid-phase synthesis. The oligomers were annealed pairwise and converted into double stranded DNA fragments by DNA polymerase I mediated repair synthesis. The fragments were assembled by ligation, and the synthetic gene was expressed in high yield in E. coli under the transcriptional control of the E. coli tryptophan promoter. The expression product was purified to homogeneity and was shown to have similar physicochemical and identical biological properties as the authentic protein isolated from the leech. Images

Rink, H; Liersch, M; Sieber, P; Meyer, F

1984-01-01

369

Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing  

PubMed Central

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the ?- and ?-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). ?-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.

Lagace, L.; Pitre, M.; Jacques, M.; Roy, D.

2004-01-01

370

Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA  

SciTech Connect

Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

Dybvig, K.

1981-01-01

371

Identification of Borrelia burgdorferi ospC genotypes in host tissue and feeding ticks by terminal restriction fragment length polymorphisms.  

PubMed

We developed a high-throughput method based on terminal restriction fragment length polymorphisms (T-RFLP) to identify ospC genotypes from field-collected samples of Borrelia burgdorferi. We first validated the method by analyzing B. burgdorferi ospC previously identified by sequencing. We then analyzed and compared ospC genotypes detected from ear biopsy tissue from natural populations of the white-footed mouse, a major B. burgdorferi reservoir host species in the eastern United States, and larval ticks feeding on those individual mice. The T-RFLP method enabled us to distinguish all 17 ospC genotypes tested, as well as mixed samples containing more than one genotype. Analysis costs compare favorably to those of alternative ospC identification methods. The T-RFLP method will facilitate large-scale field studies to advance our understanding of genotype-specific transmission patterns. PMID:23183976

Tsao, Kimberly; Bent, Stephen J; Fish, Durland

2012-11-26

372

A germline restricted, highly repetitive DNA sequence in Paramyxineatami : an interspecifically conserved, but somatically eliminated, element  

Microsoft Academic Search

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive\\u000a DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese\\u000a hagfish, Paramyxine atami. The repeat unit

S. Kubota; T. Ishibashi; S. Kohno

1997-01-01

373

Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites  

Microsoft Academic Search

RESTRICTION endonucleases recognise specific sequences in DNA, and these endonucleases, especially those which generate cohesive ends, have been widely used to clone DNA1. However, many DNAs lack sequences which are recognised by endonucleases such as EcoRI, HindIII or BamHI. A general method of overcoming this problem has been described recently2. This approach involves the synthesis of oligonucleotides sensitive to a

Bruno Gronenborn; JOACHIM MESSING

1978-01-01

374

Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids  

Microsoft Academic Search

The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm-3 and size of approximately 156 kbp are similar to

Barbara Schiller; R. G. Herrmann; G. Melchers

1982-01-01

375

Strain differentiation of epidemic typhus rickettsiae ( Rickettsia prowazekii ) by DNA restriction endonuclease analysis  

Microsoft Academic Search

DNA from five isolates ofRickettsia prowazekii of diverse origin were analyzed by restriction endonuclease digestion followed by electrophoresis. Reliable differentiation between two human isolates was possible withSacI endonuclease digestion. DNA patterns from two strains ofR. prowazekii isolated from North American flying squirrels and one strain isolated from a cattle tick were remarkably similar to those from the human isolates, whereas

Russell L. Regnery; Theodore Tzianabos; Joseph J. Esposito; Joseph E. McDade

1983-01-01

376

Efficient tracing of global isolates of Yersinia pestis by restriction fragment length polymorphism analysis using three insertion sequences as probes.  

PubMed

Yersinia pestis is the etiologic agent of plague, a disease that is transmitted from rodent to rodent and from rodent to humans by fleabites. Multiple copies of three insertion sequences (IS100, IS285, and IS1541) are scattered over the Y. pestis genome. The genomic instability generated by these insertion sequences (IS) creates a polymorphism of the hybridizing restriction fragments (restriction fragment length polymorphism [RFLP]) which can be used to subtype this relatively clonal species. The aim of this work was to evaluate and compare the potential of the three IS-RFLP techniques, individually or in combination, to define clusters of strains according to their focus of origin. The analysis of 61 Y. pestis isolates of worldwide origin indicated that no satisfactory strain clustering was observed with each IS-RFLP used individually. In contrast, the combination of the three IS-RFLP data (3IS-RFLP) resulted in both an efficient strain discrimination (D = 0.999) and a robust clustering of the isolates according to their biovar and geographical origin. This geographical clustering was observed even within the Orientalis group, although these strains had only a short period of time (one century) to diverge from the original clone that spread globally. Therefore, 3IS-RFLP is a technique that may be useful for addressing epidemiological problems and forensic issues. When plague reemerges after several decades of silence in a quiescent focus, it may help in determining whether the disease was reimported or reactivated. It may also be of value to identify the origin of a strain when plague cases appear in a previously plague-free region. Finally, this technique could be useful for the tracing of a Y. pestis isolate that has been used as a biological terrorism threat. PMID:16757602

Torrea, Gabriela; Chenal-Francisque, Viviane; Leclercq, Alexandre; Carniel, Elisabeth

2006-06-01

377

Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore  

PubMed Central

Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg2+ ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI–DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments.

Dorvel, B.; Sigalov, G.; Zhao, Q.; Comer, J.; Dimitrov, V.; Mirsaidov, U.; Aksimentiev, A.; Timp, G.

2009-01-01

378

A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.  

PubMed Central

Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described. Images

Higuchi, R; Krummel, B; Saiki, R K

1988-01-01

379

Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.  

PubMed Central

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2. Figure 3. Figure 7.

Manning, F C; Blankenship, L J; Wise, J P; Xu, J; Bridgewater, L C; Patierno, S R

1994-01-01

380

Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis.  

PubMed

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. PMID:22768309

Ambur, Ole Herman; Frye, Stephan A; Nilsen, Mariann; Hovland, Eirik; Třnjum, Tone

2012-07-02

381

Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis  

PubMed Central

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination.

Ambur, Ole Herman; Frye, Stephan A.; Nilsen, Mariann; Hovland, Eirik; T?njum, Tone

2012-01-01

382

Molecular characterization of human rotavirus VP4 genes by polymerase chain reaction and restriction fragment length polymorphism assay.  

PubMed

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups. PMID:7903789

Iizuka, M; Chiba, M; Masamune, O; Gerna, G; Nakagomi, O

1993-01-01

383

Thermodynamic DNA Looping by a Two-Site Restriction Endonuclease Studied using Optical Tweezers  

NASA Astrophysics Data System (ADS)

Many enzyme-DNA interactions involve multimeric protein complexes that bind at two distant sites such that the DNA is looped. An example is the type IIe restriction enzyme Sau3AI, which requires two recognition sites to cleave the DNA. Here we study this process at the single DNA level using force measuring optical tweezers. We characterize cleavage rates of single DNA molecules in the presence of Sau3AI as a function of enzyme concentration, incubation time, and the fractional extension of the DNA molecule. Activity is completely inhibited by tensions of a few picoNewtons. By replacing Mg^2+ with Ca^2+, the Sau3AI dimers form but do not cleave the DNA, thus trapping DNA loops. We are able to pull apart these loops, measuring the force needed and the length of DNA released for each. We also characterize the number and length distributions of these loops as a function of incubation time and DNA fractional extension. The results of these studies are discussed in the context of a Brownian dynamics model of DNA looping.

Gemmen, Gregory J.

2005-03-01

384

SURVEY AND SUMMARY: A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes  

PubMed Central

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Roberts, Richard J.; Belfort, Marlene; Bestor, Timothy; Bhagwat, Ashok S.; Bickle, Thomas A.; Bitinaite, Jurate; Blumenthal, Robert M.; Degtyarev, Sergey Kh.; Dryden, David T. F.; Dybvig, Kevin; Firman, Keith; Gromova, Elizaveta S.; Gumport, Richard I.; Halford, Stephen E.; Hattman, Stanley; Heitman, Joseph; Hornby, David P.; Janulaitis, Arvydas; Jeltsch, Albert; Josephsen, Jytte; Kiss, Antal; Klaenhammer, Todd R.; Kobayashi, Ichizo; Kong, Huimin; Kruger, Detlev H.; Lacks, Sanford; Marinus, Martin G.; Miyahara, Michiko; Morgan, Richard D.; Murray, Noreen E.; Nagaraja, Valakunja; Piekarowicz, Andrzej; Pingoud, Alfred; Raleigh, Elisabeth; Rao, Desirazu N.; Reich, Norbert; Repin, Vladimir E.; Selker, Eric U.; Shaw, Pang-Chui; Stein, Daniel C.; Stoddard, Barry L.; Szybalski, Waclaw; Trautner, Thomas A.; Van Etten, James L.; Vitor, Jorge M. B.; Wilson, Geoffrey G.; Xu, Shuang-yong

2003-01-01

385

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.  

PubMed Central

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.

Rudd, K E; Miller, W; Ostell, J; Benson, D A

1990-01-01

386

Molecular Discrimination of Lactobacilli Used as Starter and Probiotic Cultures by Amplified Ribosomal DNA Restriction Analysis  

Microsoft Academic Search

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction\\u000a analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by

Denis Roy; Stéphane Sirois; Daniel Vincent

2001-01-01

387

The migration of mitochondrial DNA fragments to the nucleus affects the chronological aging process of Saccharomyces cerevisiae  

PubMed Central

Summary Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister-Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological life span (CLS) is determined by the survival of non-dividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age.

Cheng, Xin; Ivessa, Andreas S.

2012-01-01

388

The migration of mitochondrial DNA fragments to the nucleus affects the chronological aging process of Saccharomyces cerevisiae.  

PubMed

Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister-Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological lifespan (CLS) is determined by the survival of nondividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age. PMID:20626726

Cheng, Xin; Ivessa, Andreas S

2010-10-01

389

Induction of Tumor Suppressor p53 and DNA Fragmentation in Organotypic Hippocampal Cultures Following Excitotoxin Treatment  

Microsoft Academic Search

The p53 tumor suppressor gene encodes a cell cycle regulatory protein that is induced by DNA damage and has been implicated in apoptosis. To investigate whether excitotoxic cell death due to kainic acid (KA) and cell death due toN-methyl-d-aspartate (NMDA) share similar molecular mechanisms, we studied p53 expression and DNA fragmentation in organotypic hippocampal slice cultures following excitotoxin treatment. Cellular

Shahin Sakhi; Annadora Bruce; Ning Sun; Georges Tocco; Michel Baudry; Steven S. Schreiber

1997-01-01

390

Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP  

Microsoft Academic Search

The 3.3-Ĺ resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme

D. L. Ollis; P. Brick; R. Hamlin; N. G. Xuong; T. A. Steitz

1985-01-01

391

Identification of viral proteins encoded by two DNA fragments of herpesvirus of turkeys (HVT)  

Microsoft Academic Search

Herpesvirus of turkeys (HVT) vaccine is used worldwide to immunize chickens against Marek's disease (MD). Polyclonal antiserum directed against one virus cross-reacts with proteins of the other, while only 5% homology at the DNA level was demonstrated between the two viruses. A partial library of HVT DNA fragments ranging from 1.5 to 13.5 kbp in size was constructed in pBR

H. Levy; T. Maray; I. Davidson; M. Malkinson; Y. Becker

1991-01-01

392

Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis.  

PubMed

Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results. PMID:22080038

Pascoal, Ananias; Ortea, Ignacio; Gallardo, José M; Cańas, Benito; Barros-Velázquez, Jorge; Calo-Mata, Pilar

2011-10-22

393

Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media.  

PubMed Central

A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species. These isolates were characterized by their morphologic and biochemical characteristics. Two of the isolates were identified as M. terrae complex and M. gordonae. The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp. most closely resembling M. chelonae. PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP. Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate. Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm. PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics.

Taylor, T B; Patterson, C; Hale, Y; Safranek, W W

1997-01-01

394

IS1311 and IS1245 Restriction Fragment Length Polymorphism Analyses, Serotypes, and Drug Susceptibilities of Mycobacterium avium Complex Isolates Obtained from a Human Immunodeficiency Virus-Negative Patient  

PubMed Central

Six isolates of Mycobacterium avium of genotype dnaJ+ IS901? IS1311+ IS1245+ and serotypes 6 (n = 1), 6/9, (n = 2), and 9 (n = 3) were obtained within a 5-month period from a human immunodeficiency virus-negative patient treated for tuberculosis. The isolates were identified with PvuII restriction fragment length polymorphism (RFLP) analysis as a single IS1311 RFLP type and six different IS1245 RFLP types. Six separate colonies/clones obtained by subculture from each of the six isolates were tested for MICs of a set of 10 drugs. This report documents the appearance of isolates that are resistant to antimycobacterial drugs as the duration of therapy increases. Because isolates recovered from the patient following longer duration of treatment were more likely to be resistant to more antimycobacterial drugs, we would conclude that there was selection for antimycobacterial drug-resistant isolates. Analyses of all 36 clones identified three IS1311 and 22 IS1245 types forming three clusters. Tests of 105 environmental samples collected in the home and the work place of the patient yielded 16 mycobacterial isolates, of which one M. avium from soil was of genotype dnaJ+ IS901+ IS1311+ IS1245+ and serotype 2, and the second M. avium from a vacuum cleaner was of genotype dnaJ+ IS901? IS1311+ IS1245+ and serotype 9. Overall analyses of the results did not reveal any relation between serotype, RFLP type, and drug susceptibility. Based on the course of the disease in the patient and different serotypes, IS1311 and IS1245 RFLP types of isolates of M. avium we suppose represent polyclonal infection.

Dvorska, Lenka; Bartos, Milan; Ostadal, Oldrich; Kaustova, Jarmila; Matlova, Ludmila; Pavlik, Ivo

2002-01-01

395

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.  

PubMed

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy. PMID:3912509

Warwicker, J; Ollis, D; Richards, F M; Steitz, T A

1985-12-01

396

Age- and calorie restriction-related changes in rat brain mitochondrial DNA and TFAM binding.  

PubMed

Aging markedly affects mitochondrial biogenesis and functions particularly in tissues highly dependent on the organelle's bioenergetics capability such as the brain's frontal cortex. Calorie restriction (CR) diet is, so far, the only intervention able to delay or prevent the onset of several age-related alterations in different organisms. We determined the contents of mitochondrial transcription factor A (TFAM), mitochondrial DNA (mtDNA), and the 4.8-kb mtDNA deletion in the frontal cortex from young (6-month-old) and aged (26-month-old), ad libitum-fed (AL) and calorie-restricted (CR), rats. We found a 70 % increase in TFAM amount, a 25 % loss in mtDNA content, and a 35 % increase in the 4.8-kb deletion content in the aged AL animals with respect to the young rats. TFAM-specific binding to six mtDNA regions was analyzed by mtDNA immunoprecipitation and semiquantitative polymerase chain reaction (PCR), showing a marked age-related decrease. Quantitative real-time PCR at two subregions involved in mtDNA replication demonstrated, in aged AL rats, a remarkable decrease (60-70 %) of TFAM-bound mtDNA. The decreased TFAM binding is a novel finding that may explain the mtDNA loss in spite of the compensatory TFAM increased amount. In aged CR rats, TFAM amount increased and mtDNA content decreased with respect to young rats' values, but the extent of the changes was smaller than in aged AL rats. Attenuation of the age-related effects due to the diet in the CR animals was further evidenced by the unchanged content of the 4.8-kb deletion with respect to that of young animals and by the partial prevention of the age-related decrease in TFAM binding to mtDNA. PMID:22945739

Picca, Anna; Fracasso, Flavio; Pesce, Vito; Cantatore, Palmiro; Joseph, Anna-Maria; Leeuwenburgh, Christiaan; Gadaleta, Maria Nicola; Lezza, Angela Maria Serena

2012-09-04

397

Early prediction of therapy response in patients with acute myeloid leukemia by nucleosomal DNA fragments  

PubMed Central

Background Elevated levels of nucleosomal DNA fragments can be detected in plasma and sera of patients with malignant diseases. Methods We investigated the course of nucleosomal DNA, thymidine kinase, lactate dehydrogenase and leukocytes in sera of 25 patients with acute myeloid leukemia during the first cycle of induction chemotherapy and tested their power to distinguish between patients with complete remission and those with no remission. Results Almost all patients showed strongly decreasing levels of nucleosomal DNA during the first week, in some cases after initial peaks. In overall analysis of variance, DNA levels could clearly distinguish between patients with complete remission, who had higher DNA values, and those with insufficient response (p = 0.017). The area under the curve of DNA values of days 2–4 after start of therapy (AUC 2–4) discriminated between both groups with a sensitivity of 56% at a specificity of 100%. Further, pretherapeutic levels and AUC 2–4 of nucleosomal DNA correlated significantly with blast reduction after 16 days. A tendency to higher levels in patients with complete response was also found for thymidine kinase, lactate dehydrogenase and leukocytes, however the difference did not reach the level of significance (p = 0.542, p = 0.260, and p = 0.144, respectively). Conclusion Our results indicate that nucleosomal DNA fragments are valuable markers for the early prediction of therapeutic efficacy in patients with acute myeloid leukemia.

Mueller, Susanne; Holdenrieder, Stefan; Stieber, Petra; Haferlach, Torsten; Schalhorn, Andreas; Braess, Jan; Nagel, Dorothea; Seidel, Dietrich

2006-01-01

398

Ovarian Steroids Decrease DNA Fragmentation in the Serotonin Neurons of Non-Injured Rhesus Macaques  

Microsoft Academic Search

We previously found that ovarian steroids promote neuroprotection in serotonin neurons by decreasing the expression of pro-apoptotic genes and proteins in the dorsal raphe nucleus of rhesus macaques, even in the absence of overt injury. In this study, we questioned whether these actions would lead to a reduction in DNA fragmentation in serotonin neurons. Ovariectomized (OVX) rhesus monkeys were implanted

F B Lima; C L Bethea

2010-01-01

399

Influence of PMMA shielding on DNA fragmentation induced in human fibroblasts by iron and titanium ions.  

PubMed

In the framework of a collaborative project on the influence of the shielding on the biological effectiveness of space radiation, we studied DNA fragmentation induced by 1 GeV/nucleon iron ions and titanium ions with and without a 197-mm-thick polymethylmethacrylate (PMMA) shield in AG1522 human fibroblasts. Pulsed- and constant-field gel electrophoresis were used to analyze DNA fragmentation in the size range 1-5700 kbp. The results show that, mainly owing to a higher production of small fragments (1-23 kbp), titanium ions are more effective than iron ions at inducing DNA double-strand breaks (DSBs), their RBE being 2.4 and 1.5, respectively. The insertion of a PMMA shield decreases DNA breakage, with shielding protection factors (ratio of the unshielded/shielded cross sections for DSB production) of about 1.6 for iron ions and 2.1 for titanium ions. However, the DSB yield (no. of DSBs per unit mass per unit dose) is almost unaffected by the presence of the shield, and the relative contributions of the fragments in the different size ranges are almost the same with or without shielding. This indicates that, under our conditions, the effect of shielding is mainly to reduce the dose per unit incident fluence, leaving radiation quality practically unaffected. PMID:16187791

Dini, Valentina; Antonelli, Francesca; Belli, Mauro; Campa, Alessandro; Esposito, Giuseppe; Simone, Giustina; Sorrentino, Eugenio; Tabocchini, Maria Antonella

2005-10-01

400

Preparation of DNA and RNA Fragments Containing Guanine N2-Thioalkyl Tethers  

PubMed Central

This unit describes procedures for preparation of deoxyguanosine and guanosine derivatives in which the guanine N2 contains a thiopropyl tether, protected as a tert-butyl disulfide. After incorporation into a DNA or RNA fragment, this tether allows site-specific crosslinking to a thiol of a protein or another nucleic acid.

Hou, Xiaorong; Wang, Gang; Gaffney, Barbara L.; Jones, Roger A.

2010-01-01

401

Anatomical studies of DNA fragmentation in rat brain after systemic kainate administration  

Microsoft Academic Search

Rats treated systemically with kainate develop stereotyped epileptic seizures involving mainly limbic structures that may last for hours. This model of limbic status epilepticus has been widely studied using classical neuropathological techniques. We used in situ nick translation histochemistry to examine patterns of DNA fragmentation in this model. We found a stereotyped and reproducible pattern of neuronal populations that demonstrate

S. WEISS; O. CATALTEPE; A. J. COLE

1996-01-01

402

A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea  

Microsoft Academic Search

Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans

Paula M. A. Lucchesi; Alberto E. Parma

1999-01-01

403

Formation of Apoptotic Bodies Is Associated with Internucleosomal DNA Fragmentation during Drug-Induced Apoptosis  

Microsoft Academic Search

The onset of apoptosis is often coincident with internucleosomal DNA fragmentation or ladders which are considered a hallmark of the process. However, several studies have indicated that MOLT-4 human lymphoblastoid cells exposed to various agents, including VP16, display some apoptotic characteristics in the absence of either internucleosomal ladders or production of apoptotic bodies. The present study records that, in the

Daniel R. Catchpoole; Bernard W. Stewart

1995-01-01

404

Differentiation of vaccine virus from field isolates of feline panleukopenia virus by polymerase chain reaction and restriction fragment length polymorphism analysis  

Microsoft Academic Search

In an attempt to distinguish feline panleukopenia virus (FPLV) live vaccine strains from FPLV field isolates in Japan, we compared restriction fragment length polymorphisms (RFLP) of polymerase chain reaction (PCR)-amplified fragments of live FPLV vaccine strains with those of FPLV Japanese field isolates. On the basis of nucleotide sequence differences between PLI-IV, a live vaccine strain, and FPV-483, a recent

Motohiro Horiuchi; Kazuyo Yuri; Takehisa Soma; Hiromi Katae; Hideyuki Nagasawa; Morikazu Shinagawa

1996-01-01

405

Adaptive weighted least squares method for the estimation of DNA fragment lengths from agarose gels.  

PubMed

The size of DNA fragments is most frequently estimated from their electrophoretic mobilities. Agarose gels are used to estimate the size of DNA fragments ranging from a few hundred nucleotides to more than 20 kbp. The common practice when estimating the unknown fragment sizes is to plot the log of the size of molecular weight standards against their mobility and read the values of unknowns from this graph. However, due to perturbations in the gel, such plots often show pronounced curvature which may introduce significant subjectivity into the interpolation process. We present a new method "adaptive weighted least squares (AWLS)" based on the significance test to choose the order of polynomial. We compare this with the method introduced by Schaffer based on the modification of the Southern method. The results obtained by AWLS are significantly better than the method introduced by Schaffer. Different lanes are tested for consistency. PMID:11840520

Akbari, Akbar; Albregtsen, Fritz; Lingjaerde, Ole Christian

2002-01-01

406

Statistical analyses of counts and distributions of restriction sites in DNA sequences.  

PubMed Central

Counts and spacings of all 4- and 6-bp palindromes in DNA sequences from a broad range of organisms were investigated. Both 4- and 6-bp average palindrome counts were significantly low in all bacteriophages except one, probably as a means of avoiding restriction enzyme cleavage. The exception, T4 of normal 4- and 6-palindrome counts, putatively derives protection from modification of cytosine to hydroxymethylcytosine plus glycosylation. The counts and distributions of 4-bp and of 6-bp restriction sites in bacterial species are variable. Bacterial cells with multiple restriction systems for 4-bp or 6-bp target specificities are low in aggregate 4- or 6-bp palindrome counts/kb, respectively, but bacterial cells lacking exact 4-cutter enzymes generally show normal or high counts of 4-bp palindromes when compared with random control sequences of comparable nucleotide frequencies. For example, E. coli, apparently without an exact 4-bp target restriction endonuclease (see text), contains normal aggregate 4-palindrome counts/kb, while B. subtilis, which abounds with 4-bp restriction systems, shows a significant under-representation of 4-palindrome counts. Both E. coli and B. subtilis have many 6-bp restriction enzymes and concomitantly diminished aggregate 6-palindrome counts/kb. Eukaryote, viral, and organelle sequences generally have aggregate 4- and 6-palindromic counts/kb in the normal range. Interpretations of these results are given in terms of restriction/methylation regimes, recombination and transcription processes, and possible structural and regulatory roles of 4- and 6-bp palindromes.

Karlin, S; Burge, C; Campbell, A M

1992-01-01

407

CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye.  

PubMed

Nowadays, new solutions focused on the replacement of reagents hazardous to human health are highly demanded in laboratories and Green Chemistry. In the present work, GelGreen, a new nonhazardous DNA staining reagent, has been assayed for the first time to analyze double-stranded DNA by CGE with LIF detection. The effect of GelGreen concentration on S/N ratio and migration time of a wide concentration range of standard DNA mixtures was evaluated. Under optimum GelGreen concentration in the sieving buffer efficient and sensitive separations of DNA fragments with sizes from 100-500 base pairs (bp) were obtained. A comparison in terms of resolution, time of analysis, LOD, LOQ, reproducibility, sizing performance, and cost of analysis was established between two optimized CGE-LIF protocols for DNA analysis, one based on the dye YOPRO-1 (typically used for CGE-LIF of DNA fragments) and another one using the new GelGreen. Analyses using YOPRO-1 were faster than those using GelGreen (ca. 31 min versus 34 min for the analysis of 100-500 bp DNA fragments). On the other side, sensitivity using GelGreen was twofold higher than that using YOPRO-1. The cost of analysis was significantly cheaper (ninefold) using GelGreen than with YOPRO-1. The resolution values and sizing performance were not significantly different between the two dyes (e.g. both dyes allowed the separation of fragments differing in only 2 bp in the 100-200 bp range). The usefulness of the separation method using GelGreen is demonstrated by the characterization of different amplicons obtained by PCR. PMID:23417332

Valdés, Alberto; García-Cańas, Virginia; Cifuentes, Alejandro

2013-04-23

408

Non-specific binding of restriction endonuclease EcoR1 to DNA.  

PubMed Central

Restriction endonuclease, EcoRl cleaves the DNA sequence (see formula in text) at the points indicated. Under certain conditions, EcoRl activity is observed when (see formula in text) is cut. Mg2+ is required for both activities. We find that in addition to binding to the above sites, EcoRl will also bind, although less strongly, to DNA containing neither site. Methyl acetimidate, which reacts specifically with lysine residues, inactivates the enzyme. This specific effect can be prevented by SV40 DNA and lambda DNA which contain EcoRl and EcoRl sites, by 0X174 DNA, which has only EcoRl sites and also by Polyd(AT) and polyd(GC) containing neither site. Protection occurs in the absence or presence of magnesium. The significance of this non-specific binding, both for the use and mechanism of EcoRl will be discussed.

Woodhead, J L; Malcolm, A D

1980-01-01

409

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments  

Microsoft Academic Search

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its

Fredrik Dahl; Mats Gullberg; Johan Stenberg; Ulf Landegren; Mats Nilsson

2005-01-01

410

A polymerase chain reaction-restriction fragment length polymorphism method for screening ZNF804A gene polymorphism (rs1344706) in patients with schizophrenia: a significant association.  

PubMed

The original ZNF804A rs1344706 risk variant was identified through genome-wide association studies as a risk factor for schizophrenia. Follow-up studies involving meta-analysis have confirmed that rs1344706 is a risk factor for schizophrenia as well as bipolar disorders. We describe here a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to genotype ZNF804A rs1344706 variant in patients with schizophrenia. We generated a 220 bp fragment through PCR and subsequently cleaved it by the restriction endonuclease BsaBI, creating two fragments of 114 and 106 bp. Upon change in the nucleotide from T to G, the 106 bp fragment is further cleaved by BsaBI, thus creating two fragments of 87 and 19 bp. As a result, when the 220 bp fragment is cleaved by BsaBI restriction endonuclease, the TT genotype yields two fragments of 114 and 106 bp, and TG genotype four fragments of 114, 106, 87, and 19 bp, and the GG genotype three fragments of 114, 87, and 19 bp. Thus, this is a simple, fast, and cost-effective method to genotype the ZNF804A rs1344706 risk variant. Using this method, we were able to replicate an association between ZNF804A rs1344706 variant and schizophrenia in a Turkish population. Stratification analysis of the population according to the gender showed an association that was statistically significant among overall schizophrenia and male schizophrenia and the risk T allele and TT genotype of the ZNF804A gene. PMID:21988329

Sazci, Ali; Ozel, Mavi Deniz; Ergul, Emel; Yildiz, Mustafa

2011-10-11

411

Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications  

Microsoft Academic Search

Type I restriction-modification enzymes are differentiated from type II and type III enzymes by their recognition of two specific dsDNA sequences separated by a given spacer and cleaving DNA randomly away from the recognition sites. They are oligomeric proteins formed by three subunits: a specificity subunit, a methylation subunit, and a restriction subunit. We solved the crystal structure of a

Jeong-Sun Kim; Andy Degiovanni; Jaru Jancarik; Paul D. Adams; Hisao Yokota; Rosalind Kim; Sung-Hou Kim

2005-01-01

412

A Cladistic Analysis of Phenotypic Associations With Haplotypes Inferred From Restriction Endonuclease Mapping and DNA Sequence Data. 111. Cladogram Estimation  

Microsoft Academic Search

We previously developed a cladistic approach to identify subsets of haplotypes defined by restriction endonuclease mapping or DNA sequencing that are associated with significant phenotypic deviations. Our approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram can be constructed from the restriction site or sequence data that represents the evolution- ary steps

Alan R. Templeton; Keith A. Crandall; Charles F. Sing

413

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway  

PubMed Central

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins.

van den Broek, Bram; Noom, Maarten C.; Wuite, Gijs J. L.

2005-01-01

414

Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells.  

PubMed

Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues. PMID:23203048

González-Marín, Clara; Gosálvez, Jaime; Roy, Rosa

2012-10-31

415

A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment.  

PubMed

The design of the PCR system presented in this work is based on the knowledge of the molecular character of the crown gall disease. The virulence of Agrobacterium tumefaciens requires the presence of a big (up to 235,000 bp) plasmid Ti (pTi-tumour inducing plasmid). This plasmid carries the so-called T-DNA fragment (T-DNA-transferred DNA), which integrates into cell chromosomes of the infected plants and subsequently changes the plant morphology nad metabolism. In cannot be excluded that after T-DNA integration the presence of Agrobacterium is not necessary for the development of pathological changes. Thus, T-DNA is the only sign that must be present both in virulent bacteria and in infected plants in any stadium of the disease and even before the infection. This is why T-DNA was chosen as the target region for PCR amplification. Primers flanking a 220 bp fragment of one of the conservative regions responsible for Agrobacterium pathogenicity, namely tms2 gene coding for indolacetamide amidohydrolase (the second step of auxin biosynthesis) were designed as the optimal for PCR amplification. The PCR amplification reactions were performed for matrixes isolated from cultures of reference strains giving one predicted product for each sample. First attempts of T-DNA detection in infected soils and plants were performed. We hope that the presented new PCR system for Agrobacterium tumefaciens detection will help to fight the crown gall disease in the nearest future. PMID:9429288

Sachadyn, P; Kur, J

1997-01-01

416

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.  

PubMed

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp. PMID:24019490

Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

2013-09-09

417

Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.  

PubMed Central

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication. Images

van Bergen, B G; van der Ley, P A; van Driel, W; van Mansfeld, A D; van der Vliet, P C

1983-01-01

418

Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes.  

PubMed

We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assays results in a real cloning experience, we performed a cloning simulation assay using the same conditions established for the radioactive assay (enzyme units and pmols of DNA ends). As a result, we found that for degradation percentages of the radioactive DNA substrate per enzyme unit below 0.5, the false positives in the cloning stimulation assay were less than 5%. This conditions could ensure a good performance of the enzyme preparations for cloning experiments. Finally, we described the use of the radiolabeled [gamma 33P] ATP lambda Hpa II DNA substrate to detect 5'-->3' single stranded DNA dependent exonuclease and phosphatase contaminating activities in some critical steps of the purification process of the restriction enzyme Kpn I. PMID:8783903

Trujillo, L E; Pupo, E; Miranda, F; Pérez, E; González, E

419

The analysis of groEL gene in Salmonella enterica subspecies enterica serovar Typhimurium isolated from avians by PCR-Restriction Fragment Length Polymorphism method.  

PubMed

Salmonella enterica subspecies enterica serovar Typhimurium causes food-borne outbreaks and systemic diseases in humans and animals. groEL gene (also known as mopA gene in S. Typhimurium), possessing conserved sequence, plays an important role in invasion of bacteria. The purpose of present study was to identify the polymorphism of groEL gene among different avians in different regions by PCR-RFLP method. Fifty two S. Typhimurium isolates (Broiler (n = 13), Layer (n = 12), Duck (n = 5), Goose (n = 5), Sparrow (n = 8), Canary (n = 3), Pigeon (n = 5) and Casco parrot (n = 1). were identified using serotyping as well as multiplex-PCR. Then, amplification of groEL gene performed and amplified products subjected to restriction digestion with BsuRI enzyme. Three RFLP profiles, A, B and C, generated DNA fragments between approximately 100-1,000 bp in size, were observed. The RFLP profile A was observed in 35 (67.3%), profile B in 14 (26.9%) and profile C in 3 (5.77%) of isolates. S. Typhimurium isolates recovered from 13 broilers (two of which profile A, 9 profile B and 2 profile C) and from 8 sparrows (two of which profile A, 5 profile B and 1 profile C) showed all three profiles, but 12 layers and other avians (including Canary (n = 3), Goose (n = 5), Duck (n = 5), Pigeon (n = 5) and Casco parrot (n = 1)) showed profile A. None of these profiles was allotted for a special region. The result of present study showed that S. Typhimurium undergoes genetic mutations in groEL gene under unpleasant milieu in different regions and in different avians. Thus, genetic diversity, despite conserved nature of groEL gene in S. Typhimurium, may exist but it depends on the condition where bacteria have settled. To our knowledge, three RFLP profiles of groEL gene generated by BsuRI restriction enzyme were not reported previously. PMID:21312060

Dilmaghani, Mahdi; Ahmadi, Malahat; Zahraei Salehi, Taghi; Talebi, Alireza

2011-02-11

420

Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by sequencing and restriction fragment length polymorphism analysis of the pvpA gene.  

PubMed

Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host-parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host-parasite relationship. PMID:14562892

Pillai, S R; Mays, H L; Ley, D H; Luttrell, P; Panangala, V S; Farmer, K L; Roberts, S R

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