Immunoseparation and immunodetection of nucleic acids labeled with halogenated nucleotides.
AuthorsHaider SR, Juan G, Traganos F, Darzynkiewicz Z
JournalExp Cell Res
PubMed ID9260920
'A novel methodology for labeling, isolation, and detection of nucleic acids is described. Nucleic acid isolation is based on in vivo or in vitro incorporation of BrU or BrdU to either RNA or DNA, respectively, followed by immunoprecipitation of the labeled nucleic acid utilizing anti-BrdU MoAb, which crossreacts with BrU, ... More
Purification and characterization of human topoisomerase I mutants.
AuthorsJensen AD, Svejstrup JQ
JournalEur J Biochem
PubMed ID8612607
'A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their ... More
Flow cytometric evaluation of apoptosis, necrosis and recovery when culturing monocytes.
'After developing and applying a method for cryopreserving monocytes, we found a substantial cell loss when culturing these cells. Monocytes were isolated from blood donors by density gradient centrifugation, purified by elutriation and cryopreserved. Thawed cells were cultured in ultra low attachment wells and studied with Annexin V, Propidium iodide, ... More
A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase.
AuthorsPorstmann T, Meissner K, Glaser R, Döpel SH, Sydow G
JournalJ Virol Methods
PubMed ID1713913
'A sensitive non-isotopic assay for specific detection of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is described using 5-bromo-2''-deoxyuridine triphosphate (BrdUTP) instead of tritiated thymidine triphosphate. After the RT reaction the template primer is degraded by alkaline hydrolysis. Single-stranded poly.(BrdU) is detected in an immunoenzymometric assay ... More
A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation.
'A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridined 5''-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using ... More
Pairing properties of bromouracil and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis.
AuthorsMuller M, Martial J, Verly WG
JournalBiochem J
PubMed ID3178734
'5-Bromo-2''-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I. When DNA containing Br-dUMP instead of dTMP at a few preselected sites is transfected into competent bacteria, no mutation occurs, indicating that in vivo E. coli DNA ... More
Application of 5-bromo-2'deoxyuridine as a label for in situ hybridization in chromosome microdissection and painting, and 3' OH DNA end labeling for apoptosis.
AuthorsMühlmann-Díaz MC, Dullea RG, Bedford JS
JournalBiotechniques
PubMed ID8816240
'We have utilized 5-bromo-2''deoxyuridine (BrdU) substituted DNA as a probe for a number of applications including, principally, for chromosome painting by fluorescence in situ hybridization (FISH) but also for DNA end-labeling to detect apoptotic cell death and for filter hybridization. Br-dUTP was used as a substitute for biotin or digoxigenin-dUTP ... More
T4 phage ribonucleotide reductase. Allosteric regulation in vivo by thymidine triphosphate.
AuthorsJi JP, Sargent RG, Mathews CK
JournalJ Biol Chem
PubMed ID1885561
'Based upon analyses of purified enzyme preparations, T4 bacteriophage-coded ribonucleotide reductase is considered to be relatively insensitive to control by allosteric inhibition. However, two factors suggest that CDP reduction to dCDP is feedback-controlled by dTTP in infected cells. First, the pool of 5-hydroxymethyldeoxycytidine triphosphate, which expands manyfold upon infection by ... More
In vitro mutagenesis of Xanthomonas campestris alpha-amylase gene by partially replacing deoxythymidine triphosphate with 5-bromo-2'-deoxyuridine-5'-triphosphate using a PCR technique.
AuthorsMa XD, Ke T, Li YX, Huang CX, Song AD, Chen HG, Wu YH, Jia XC, He GY
JournalBiotechnol Lett
PubMed ID15000487
Three mutants of the wild type alpha-amylase gene from Xanthomonas campestris pv. campestris 8004 were obtained using a PCR technique in which deoxythymidine triphosphate (dTTP) was partially replaced by 5-bromo-2'-deoxyuridine-5'-triphosphate (BrdUTP), at an optimal dTTP:BrdUTP ratio of 1000:1. Of thre three mutants that were obtained and which were sequenced, one ... More
Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI.
AuthorsBodnar JW, Zempsky W, Warder D, Bergson C, Ward DC
JournalJ Biol Chem
PubMed ID6317689
The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in ... More
Cyclin-dependent kinases govern formation and maintenance of the nucleolus.
AuthorsSirri V, Hernandez-Verdun D, Roussel P
JournalJ Cell Biol
PubMed ID11901165
In higher eukaryotic cells, the nucleolus is a nuclear compartment assembled at the beginning of interphase, maintained during interphase, and disorganized during mitosis. Even if its structural organization appears to be undissociable from its function in ribosome biogenesis, the mechanisms that govern the formation and maintenance of the nucleolus are ... More
Nucleolar assembly of the rRNA processing machinery in living cells.
AuthorsSavino TM, Gébrane-Younès J, De Mey J, Sibarita JB, Hernandez-Verdun D
JournalJ Cell Biol
PubMed ID11381093
To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and ... More
Colorimetric capture assay for human-immunodeficiency-virus-I reverse transcriptase activity.
The development of a colorimetric capture assay for HIV-1 reverse transcriptase (RT) activity is described. This assay consisted of three basic steps: enzyme purification, RT reaction and product detection, which were all performed in the same microtitre plate. Mouse monoclonal anti-RT antibodies of subclass G2a were bound by polyclonal goat ... More
Ribonucleotide reductase: a determinant of 5-bromodeoxyuridine mutagenesis in phage T4.
AuthorsSargent RG, Ji JP, Mun B, Mathews CK
JournalMol Gen Genet
PubMed ID2671647
Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step. Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions. ... More
Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin.
AuthorsLorenz JD, Watkins JF, Smerdon MJ
JournalMutat Res
PubMed ID3347208
We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, ... More