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At least 127 records · Page 7Linked to original sources

Separate analysis of complementary strands of restriction enzyme-digested DNA. An application of restriction fragment mass mapping by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) of a restriction endonuclease digest determines the molecular mass of PCR-amplified DNA more easily than measurement of undigested DNA. With this method, a 664 bp region from the FAS gene could be analyzed and a two-nucleotide deletion in the L1CAM gene was detected in a restriction fragment of 105 nucleotides. Furthermore, the analysis of smaller fragments allowed separate detection of single-stranded oligonucleotides comprising individual digested fragments. This mixture analysis of restriction enzyme digests improves the resolution, sensitivity and accuracy of MALDI/TOF-MS of DNA and is thus expected to facilitate its application to genetic diagnosis.

DNA↗

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

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 (1986, Gene 41: 129-134) 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.

DNA↗

Molecular epidemiology of group B streptococcal infections: use of restriction endonuclease analysis of chromosomal DNA and DNA restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping).

Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therefore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. HhaI gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype Ia/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.

Adult↗

Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restriction fragment length polymorphism, and pulsed-field gel electrophoresis.

The genomes of 62 North American and European Borrelia burgdorferi isolates were examined by restriction endonuclease analysis (REA), gene probe restriction fragment length polymorphism, and pulsed-field gel electrophoresis (PFGE). Hybridization of restriction fragments with the immunologically relevant 83-kDa antigen gene revealed polymorphisms and divided the isolates into three major groups. Group I included all but two of the American isolates and some of the European isolates. One of two Californian isolates (DN 127) and one Ixodes dammini isolate from New York (strain 25015), previously described as atypical, were distinct from the isolates in the three groups. Plasmid profile analysis and REA, the method with the highest level of discrimination, revealed extensive heterogeneity among isolates of the same major group. Our study demonstrates the usefulness of the 83-kDa antigen gene probe for dividing the isolates into major genogroups, whereas REA and plasmid profile analysis allow for a distinction of individual strains within these groups.

Animals↗

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.

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.

Animals↗

Effects of intensive lifestyle interventions with calorie-carbohydrate-restricted diet versus time-restricted eating on appetite and binge eating in type 2 diabetes: A randomized controlled trial.

The impact of intensive lifestyle interventions on appetite regulation and binge eating in individuals with type 2 diabetes (T2D) remains unclear. This study evaluated the effects of combined lifestyle interventions on appetite responses and binge eating in overweight or obese adults with T2D. In a randomized trial, 120 participants with T2D were allocated to three groups (n&#xa0;=&#xa0;40 each): (1) Calorie-carbohydrate restriction (CCR), (2) Time-restricted eating with CCR (TRE&#xa0;+&#xa0;CCR), or (3) Control. Intervention groups received structured exercise and behavioral education based on the Information-Motivation-Behavioral Skills model. Appetite perceptions (hunger, satiety, desire to eat, and prospective food consumption) and binge eating (Binge Eating Scale; BES and objective binge episodes) were evaluated at baseline, week 12, and week 24 using linear mixed models. Both CCR and TRE&#xa0;+&#xa0;CCR significantly improved subjective appetite compared with the control group at 12 and 24 weeks (all p&#xa0;<&#xa0;0.01). At 24 weeks, hunger decreased by -24.1&#x202f;mm (95% CI: -35.8, -12.5) in the CCR group and -32.7&#x202f;mm (95% CI: -44.2, -21.3) in the TRE&#xa0;+&#xa0;CCR group. Satiety also increased by 21.7&#x202f;mm (95% CI: 9.46, 33.9) and 29.4&#x202f;mm (95% CI: 17.4, 41.4), respectively. Significant reductions were observed for desire to eat and prospective food consumption. In contrast, changes in BES and objective binge episodes were not significantly different between groups at any time point. No significant differences were detected between the CCR and the TRE&#xa0;+&#xa0;CCR groups. Intensive lifestyle interventions incorporating CCR or TRE&#xa0;+&#xa0;CCR effectively reduced appetite in adults with T2D but did not significantly affect binge eating. Future research should target individuals with higher baseline BES scores to clarify potential benefits for binge eating behavior.

Humans↗

Restriction fragment length polymorphism of the human T cell receptor alpha gene. I. Two polymorphic restriction sites localized to different regions of the gene.

Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3' joining (J) exon and the most 5' constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3' untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.

Alleles↗

Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels.

We have studied with a number of bacterial restriction enzymes the conditions for digestion of DNA in agarose and polyacrylamide gels. The restriction endonucleases HpaII, MspI, HaeIII, HindIII, TaqI, HhaI, AluI, BamHI, EcoRI and SalI are capable of digesting DNA in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI, are also capable of digesting DNA in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible.

Animals↗

Cloning and characterization of the unusual restriction-modification system comprising two restriction endonucleases and one methyltransferase.

An Escherichia coli RFL47 DNA fragment containing the Eco47IR and Eco47II restriction-modification (R-M) system has been cloned and sequenced. A clone carrying this system has been selected by its ability to restrict phage lambda in vivo. The sequence of 5360 bp was determined, and its analysis revealed three major open reading frames (ORF) corresponding to two restriction endonucleases (ENases) and one DNA methyltransferase (MTase): R.Eco47II (239 amino acid (aa)), R.Eco47I (230 aa) and M.Eco47II (417 aa). The M.Eco47II aa sequence possesses all conserved domains typical for m5C MTases and its variable region has a high homology with M.Sau96I and M.SinI. The ORF harboring a predicted helix-turn-helix motif upstream from the eco47IR gene has been found. No sequence resembling the eco47IM gene has been detected in the complete fragment sequenced, although disrupted ORF, possibly corresponding to the transposase-encoding gene, has been found in the intergenic area between eco47IIM and eco47IR. No homology was found between the ENases; however, both revealed homology with their isoschizomers, R.SinI and R.Sau96I.

Amino Acid Sequence↗

Subtracted restriction fingerprinting--a new typing technique using magnetic capture of tagged restriction fragments.

Molecular typing of bacterial pathogens is an important issue in the epidemiological analysis of emerging infections in humans and animals. Numerous methods have been developed for and applied to a wide variety of bacteria of medical, veterinary and zoonotic importance. The present minireview provides a description of a new typing approach designated subtracted restriction fingerprinting (SRF), its use for typing of Salmonella isolates and a comparison with the most widely used typing techniques for these bacteria. SRF is based on double restriction endonuclease digestion of whole cell DNA, followed by a fill-in reaction with specifically tagged nucleotides and subtractive capture of selected restriction fragments. This results in a reduced number of fragments optimal for separation in standard agarose gels.

Animals↗

PspGI, a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes.

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.

Amino Acid Sequence↗

An immunodominant class I-restricted cytotoxic T lymphocyte determinant of human immunodeficiency virus type 1 induces CD4 class II-restricted help for itself.

We have observed that a peptide corresponding to an immunodominant epitope of the HIV-1 envelope protein recognized by class I MHC-restricted CD8+ CTL can also induce T cell help for itself. The help is necessary for restimulation of CTL precursors in vitro with peptide alone in the absence of exogenous lymphokines, can be removed by depletion of CD4+ T cells, and can be replaced by exogenous IL-2. Whereas the CTL in BALB/c or B10. D2 mice are restricted by the class I molecule Dd, the Th cells are restricted by the class II molecule Ad, and the help can be blocked by anti-Ad mAb. To examine the genetic regulation of the induction of help, we studied B10.A mice that share the class I Dd molecule, but have different class II molecules, Ak and Ek. Spleen cells of immune B10.A mice behave like CD4-depleted BALB/c spleen cells in that they cannot be restimulated in vitro by the peptide alone, but can with peptide plus IL-2. Therefore, in the absence of exogenous lymphokines, peptide-specific help is necessary for restimulation with this immunodominant CTL epitope peptide, and in H-2d mice, this peptide stimulates help for itself as well as CTL. We speculate on the implications of these findings for the immunodominance of this peptide in H-2d mice, and for the selective advantage of pairing certain class I and class II molecules in an MHC haplotype.

Animals↗

Plasmid R16 ArdA protein preferentially targets restriction activity of the type I restriction-modification system EcoKI.

The ArdA antirestriction protein of the IncB plasmid R16 selectively inhibited the restriction activity of EcoKI, leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The results are consistent with the hypothesis that ArdA functions in bacterial conjugation to allow an unmodified plasmid to evade restriction in the recipient bacterium and yet acquire cognate modification.

Bacterial Proteins↗

Characterization of noncapsulate Haemophilus influenzae by whole-cell polypeptide profiles, restriction endonuclease analysis, and rRNA gene restriction patterns.

Thirty-four clinical isolates of noncapsulate Haemophilus influenzae representing isolates with either related or dissimilar patterns of whole-cell polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were further characterized by restriction enzyme analysis (REA) and rRNA gene restriction patterns. Total cellular DNA was extracted by a rapid, microcentrifuge-scale method and digested with BamHI, which gave a pattern of about 18 discrete bands. This confirmed the five closely related groupings suggested by SDS-PAGE. Isolates dissimilar by SDS-PAGE were also distinguishable by REA. However, there was no correlation between the degrees of similarity estimated from whole-cell polypeptide profiles and those obtained from REA for the dissimilar isolates. Therefore, inferences of genetic relatedness made on the basis of these data should be interpreted with caution. rRNA gene restriction patterns also confirmed the groupings suggested by the other two techniques. We conclude that the three methods were highly discriminatory and that whole-cell polypeptide patterns or REA with BamHI would be appropriate techniques for epidemiological studies of noncapsulate H. influenzae.

Bacterial Proteins↗

Hae III--a suitable restriction endonuclease for restriction fragment length polymorphism analysis of biological evidence samples.

Hae III has been selected by our laboratories as the restriction endonuclease of choice for restriction fragment length polymorphism analysis of forensic science samples. The enzyme is compatible with the D2S44 probe system and generates relatively small DNA fragments for that marker system. Similarly, Hae III is compatible with several other independent polymorphic loci, including D1S7, D4S139, D16S85, D17S74, D17S79, D14S13, and D20S15. Hae III is functional under a variety of adverse conditions for DNA digestion and is not affected by the methylation pattern in mammals. Finally, Hae III is a relatively inexpensive restriction endonuclease.

Base Sequence↗

Analysis of left ventricular diastolic function by the pressure-volume relation in cases with idiopathic restrictive cardiomyopathy and amyloid restrictive cardiomyopathy.

To assess left ventricular (LV) diastolic function in idiopathic restrictive cardiomyopathy (IRCM) and amyloid restrictive cardiomyopathy (ARCM), we analyzed LV cineangiograms and pressures (tip manometer) in two patients, one with IRCM and the other with ARCM. LV volume and instantaneous rate of change of volume were derived from frame-by-frame analyses of angiograms. LV relaxation was assessed by the time constant of isovolumic pressure decay. LV compliance was evaluated by the pressure-volume relationship. From these analyses, we conclude as follows: (1) In restrictive cardiomyopathy (RCM), LV diastolic dysfunction may be characterized by impaired relaxation and decreased compliance; (2) LV diastolic filling was minimally impaired or normal in both conditions; (3) The ventricular diastolic pressure wave form of "dip and plateau" seemed not to be a characteristic sign in RCM.

Blood Pressure↗

Increased frequency of 2,4,6-trinitrophenyl (TNP)-specific, H-2b-restricted cytotoxic T lymphocyte precursors in transgenic mice expressing a T cell receptor beta chain gene from an H-2b-restricted, TNP-specific cytolytic T cell clone.

T cell antigen receptors (TcR) expressing V alpha 10/J alpha BBM142 genes in association with beta chains containing J beta 2.6 elements were found to be predominant among 2,4,6-trinitrophenyl (TNP)-specific, H-2b-restricted cytolytic T cell lines (CTL). To assess the relative contribution of the TcR beta chain to the TNP specificity as well as to the selection of the respective TcR alpha chain elements we generated transgenic mice expressing the TcR beta chain of the H-2b/TNP-specific CTL clone BT7.4.1. The TcR of this clone does not belong to the type predominant among H-2b/TNP-specific CTL, as it consists of an alpha chain encoded by a V alpha 8/J alpha DO gene rearrangement and a V beta 2/J beta 1.1-containing beta chain. In the transgenic mice almost all T cells exclusively express the transgenic V beta 2 gene, as a result of allelic exclusion. TNP-specific, H-2b-restricted precursors were found at 7- to 8-fold higher frequency in these mice as compared with non-transgenic littermates. In H-2b/d heterozygous transgenic mice, an increased frequency of TNP-specific precursors was found only in H-2b, but not in H-2d-restricted CTL. Analysis of H-2b/TNP-specific CTL lines derived from V beta 2-transgenic mice indicated a preferential association of the transgenic TcR beta chain with endogenous alpha chains encoded by V alpha 8 and J alpha BBM142 genes. This suggests that the hapten TNP is recognized like typical peptide antigens by combinatorial TcR alpha and beta contact sites.

Alleles↗