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Journal of Bacteriology Jun 1988By random transposon Tn5 insertions, we previously identified six virulence-associated SalI fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of...
By random transposon Tn5 insertions, we previously identified six virulence-associated SalI fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigella flexneri 2a. In this study, we analyzed the sites of 134 independent Tn5 insertions on four contiguous SalI fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichia coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for full expression of the virulence phenotype of shigellae.
Topics: DNA Restriction Enzymes; DNA Transposable Elements; DNA, Bacterial; Deoxyribonuclease EcoRI; Deoxyribonucleases, Type II Site-Specific; Nucleic Acid Hybridization; Phenotype; Plasmids; Shigella flexneri
PubMed: 2836357
DOI: 10.1128/jb.170.6.2480-2484.1988 -
The Journal of Biological Chemistry Dec 1987A cDNA clone, lambda rCB1, encoding calmodulin was isolated from a rat brain expression library. The sequence was determined and compared to the structures of the... (Comparative Study)
Comparative Study
A cDNA clone, lambda rCB1, encoding calmodulin was isolated from a rat brain expression library. The sequence was determined and compared to the structures of the previously described rat genes, lambda SC4 and lambda SC8 (Nojima, H., and Sokabe, H. (1986) J. Mol. Biol. 190, 391-400). Faithful sequence conservation is observed in the coding regions of lambda rCB1 and lambda SC4, the bona fide gene. Both cDNAs encode identical amino acid sequence. Very limited sequence homology, however, is noted in the 3'-untranslated segments of these clones. Surprisingly, when the lambda rCB1 nucleotide structure is compared to the processed intronless gene, lambda SC8, extensive sequence homology is found both in the coding and noncoding regions. The inferred protein sequences of lambda SC8 and lambda rCB1, however, are divergent. Using a fragment of lambda rCB1 to screen an expression library derived from a human embryonic cell line, a calmodulin cDNA clone, lambda hCE1, was isolated and characterized. Comparison of the sequence of lambda hCE1 to the cDNA from human liver, hCWP (Wawrzynczak, E. J., and Perham, R. N. (1984) Biochem. Int. 9, 177-185), reveals substantial structural divergence. Strikingly poor homology is seen in the 5'- and 3'-noncoding segments, but the coding regions were 85% homologous. Both lambda hCE1 and hCWP encode proteins of identical primary structure which is equivalent to the protein sequence deduced from lambda rCB1 and lambda SC4. Taken together these results suggest the existence of an additional actively transcribed calmodulin gene, not previously identified, in each of the human and rat genomes. Transcripts of lambda rCB1 and lambda hCE1 were observed in all tissues examined indicating the absence of tissue-specific expression. Calmodulin gene polymorphisms were detected using TaqI, HindIII, and MspI.
Topics: Amino Acid Sequence; Animals; Base Sequence; Calmodulin; DNA; DNA Restriction Enzymes; Deoxyribonuclease HindIII; Deoxyribonuclease HpaII; Deoxyribonucleases, Type II Site-Specific; Gene Expression Regulation; Genes; Humans; Molecular Sequence Data; Polymorphism, Genetic; Rats
PubMed: 2445749
DOI: No ID Found -
Journal of Dairy Science Jul 2019We scanned the genome of 77,815 Normande cattle with different Illumina SNP chips (Illumina Inc., San Diego, CA) to map recessive embryonic lethal mutations using...
We scanned the genome of 77,815 Normande cattle with different Illumina SNP chips (Illumina Inc., San Diego, CA) to map recessive embryonic lethal mutations using homozygous haplotype deficiency. We detected 2 novel haplotypes on chromosomes 11 and 24 but did not confirm 6 previously reported haplotypes. The one on chromosome 11 showed a marked reduction in conception rates and moderate decrease in nonreturn rate in at-risk versus control mating, supporting late embryonic mortality. After fine mapping and analyzing whole-genome sequences, we prioritized a missense mutation in CAD (g.72399397T>C; p.Tyr452Cys)-a gene encoding a protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) essential for de novo pyrimidine biosynthesis-as a candidate causal variant. This transition mutation replaces a tyrosine residue, which is perfectly conserved among living organisms, with a cysteine residue in the carbamoyl-phosphate synthetase 2 domain of the protein. A single animal was confirmed to be homozygous for the mutation based on Sanger sequencing. However, large-scale genotyping of the candidate variant with the Illumina EuroG10k BeadChip revealed an absence of live homozygotes in a panel of 33,323 Normande animals and an absence of carriers in 348,593 animals from 19 other cattle breeds. These results support recessive embryonic lethality with nearly complete penetrance, as was previously reported in CAD mutants in several eukaryote species. The only homozygous cow had extremely poor udder conformation, suggesting a potential role of CAD in udder development, but no effect was detected when comparing daughter yield deviations of 250 heterozygous bulls with that of 2,912 homozygotes for the ancestral allele. Together, our results showed the importance of large-scale screening for homozygous haplotype deficiency with hundreds of thousands of animals, validating results with an independent data set, and considering unexpected live homozygotes, to avoid both false-positive and false-negative discoveries. These discoveries will be used primarily in mating decisions to avoid at-risk mating. In addition, we recommend including CAD in the breeding objectives of Normande cattle.
Topics: Alleles; Animals; Breeding; Cattle; Deoxyribonucleases; Female; Fertilization; Haplotypes; Heterozygote; Homozygote; Male; Mutation; Mutation, Missense; Polymorphism, Single Nucleotide; Reproduction
PubMed: 31056337
DOI: 10.3168/jds.2018-16100 -
Clinical Biochemistry Oct 2015The extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present... (Comparative Study)
Comparative Study
OBJETIVES
The extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool.
DESIGN AND METHODS
EDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase.
RESULTS
The endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma.
CONCLUSION
The exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma.
Topics: Anticoagulants; Biomarkers; Calcium Chelating Agents; Chromosomes, Human, Y; DNA; Deoxyribonuclease I; Deoxyribonucleases; Edetic Acid; Enzyme Inhibitors; Female; Genetic Testing; Humans; Hydrolysis; Male; Molecular Diagnostic Techniques; Plasma; Pregnancy; Prenatal Diagnosis; Serum; Temperature
PubMed: 25746148
DOI: 10.1016/j.clinbiochem.2015.02.014 -
The Journal of Biological Chemistry Dec 1981An ATP-dependent deoxyribonuclease was isolated from lymphocyte nuclei. The enzyme preparation sediments with about 4 S through sucrose gradients and shows one stainable...
An ATP-dependent deoxyribonuclease was isolated from lymphocyte nuclei. The enzyme preparation sediments with about 4 S through sucrose gradients and shows one stainable band after sodium dodecyl sulfate gel electrophoresis. We find three, possibly four, activities associated with the enzyme: a DNA-independent ATPase activity; an ATP-independent endonuclease; an ATP-dependent nuclease which degrades nicked DNA to acid-soluble material; and an unwinding activity producing single-stranded regions in nicked DNA.
Topics: Adenosine Triphosphate; Animals; Cattle; Deoxyribonucleases; Kinetics; Lymph Nodes; Lymphocytes
PubMed: 7309758
DOI: No ID Found -
The Journal of Biological Chemistry Nov 1959
Topics: DNA; Deoxyribonucleases; Endodeoxyribonucleases; Ions; Magnesium; Sodium
PubMed: 14445348
DOI: No ID Found -
Nucleic Acids Research Oct 1997Here we report the use of exonuclease to expose complementary DNA between an insert and vector such that annealing becomes independent of restriction site compatibility....
Here we report the use of exonuclease to expose complementary DNA between an insert and vector such that annealing becomes independent of restriction site compatibility. We demonstrate that unusual and, in some cases, previously impossible cloning strategies can be readily and efficiently achieved as long as the flanking sequences of the linear vectors are highly related. Furthermore, we show that the bacterial repair system resolves the residual mismatches, overhangs or gaps in a predictable fashion to generate excisable inserts. This approach facilitates cloning regardless of restriction site compatibility and overcomes an important limitation in current cloning techniques.
Topics: Base Sequence; Binding Sites; Cloning, Molecular; DNA Restriction Enzymes; DNA, Complementary; DNA-Directed DNA Polymerase; Deoxyribonuclease EcoRI; Deoxyribonucleases, Type II Site-Specific; Exodeoxyribonucleases; Genetic Vectors; Molecular Sequence Data; Viral Proteins
PubMed: 9321675
DOI: 10.1093/nar/25.20.4165 -
The European Respiratory Journal Apr 1996Recombinant human deoxyribonuclease (rhDNase) has been demonstrated to reduce in vitro the viscosity and to improve the transport capacity of purulent respiratory mucus...
Recombinant human deoxyribonuclease (rhDNase) has been demonstrated to reduce in vitro the viscosity and to improve the transport capacity of purulent respiratory mucus in cystic fibrosis. During episodes of exacerbation of chronic bronchitis, the patients generally expectorate purulent mucus. Purulence of mucus is associated with an increased deoxyriboneucleic acid (DNA) concentration. We analyzed in vitro the potential effect of rhDNase on chronic bronchitis mucus transport by the ciliary activity (frog palate model) and by simulated cough (cough machine model), as well as the effect on mucus viscosity (controlled stress rheometer) and surface properties (contact angle). Purulent sputa collected from patients with chronic bronchitis (n = 15) during an episode of exacerbation were incubated for 30 min at 37 degrees C with either rhDNase at two different concentrations (final concentration 2 or 4 micrograms.mL-1) or placebo. The median mucociliary transport rate was significantly improved by rhDNase from 0.68 with placebo to 0.79 and 0.83 with 2 and 4 micrograms.mL-1 of rhDNase, respectively. A significant improvement in mucus cough transport was also induced by rhDNase from 25.5 mm with placebo to 27.0 mm with either 2 or 4 micrograms.mL-1 rhDNase. These improvements in mucus transport capacity were associated with alterations in the physical properties of the mucus. The mucus median control viscosity (511.4 Pa.s) and median contact angle (0.85 rd) significantly decreased to 112.5 Pa.s and 0.74 rd, respectively, in the presence of 4 micrograms.mL-1 of rhDNase. These findings demonstrate that recombinant deoxyribonuclease may exert a beneficial effect on mucus clearance in vitro by altering the viscosity and surface properties of the purulent chronic bronchitic sputum samples.
Topics: Bronchitis; Chronic Disease; Cough; DNA; Deoxyribonucleases; Female; Humans; Male; Mucociliary Clearance; Mucus; Recombinant Proteins; Surface Properties; Viscosity
PubMed: 8726943
DOI: 10.1183/09031936.96.09040769 -
Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications.Nucleic Acids Research Jan 2001We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson-Crick structural... (Comparative Study)
Comparative Study
We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson-Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert ( approximately 20 bp) containing the modification or non-Watson-Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields ( approximately 60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid-protein interactions.
Topics: Base Composition; Base Pairing; DNA; Deoxyribonuclease BamHI; Deoxyribonuclease EcoRI; Deoxyribonucleases, Type II Site-Specific; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; Polymerase Chain Reaction; RNA, Ribosomal; Ribose
PubMed: 11139636
DOI: 10.1093/nar/29.2.e6 -
Nucleic Acids Research Jun 2009The biological functions of DNA-binding proteins often require that they interact with their targets with high affinity and/or high specificity. Here, we describe a...
The biological functions of DNA-binding proteins often require that they interact with their targets with high affinity and/or high specificity. Here, we describe a computational method that estimates the extent of optimization for affinity and specificity of amino acids at a protein-DNA interface based on the crystal structure of the complex, by modeling the changes in binding-free energy associated with all individual amino acid and base substitutions at the interface. The extent to which residues are predicted to be optimal for specificity versus affinity varies within a given protein-DNA interface and between different complexes, and in many cases recapitulates previous experimental observations. The approach provides a complement to traditional methods of mutational analysis, and should be useful for rapidly formulating hypotheses about the roles of amino acid residues in protein-DNA interfaces.
Topics: Amino Acids; Computational Biology; Crystallography, X-Ray; DNA; DNA Restriction Enzymes; DNA-Binding Proteins; Deoxyribonuclease I; Deoxyribonucleases, Type II Site-Specific; Endonucleases; Models, Molecular; Transcription Factors
PubMed: 19389725
DOI: 10.1093/nar/gkp242