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At least 127 records · Page 7Linked to original sources
PubMed1999

Nucleoprotein gene tracking: localization of specific HIV-1 genes to subchromatin nucleoprotein complexes containing endonuclease activity in HIV-1-infected human cells.

We developed a technique with which to isolate specific subchromatin deoxyribonucleoprotein/ribonucleoprotein precursor complexes containing discrete genes from intact mammalian nuclei by direct restriction enzyme treatment with MspI. These nucleoprotein complexes can be further fractionated and purified by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electroelution and removal of detergent, virtually thousands of nucleoprotein complexes can be examined for the presence of tightly bound genes and enzymatic activities. Nucleoprotein gene tracking procedures were applied to study the acidic nucleoprotein complexes from steady-state human H9 cells uninfected or infected with human immunodeficiency virus type 1 (HIV-1) virus. The purified nucleoprotein complexes were screened for the presence of loosely and tightly associated HIV-1 gene sequences (pol, env, tat, and rev) using a DNA hybridization protocol. In HIV-1-infected cells, four specific nucleoprotein complexes out of several hundred were found to contain tightly bound HIV-1 pol gene sequences after purification. By contrast, the other HIV-1 gene sequences (env, tat, and rev) were not tightly bound to any of the nucleoprotein complexes in HIV-infected cells. The observations suggest that certain HIV-1 genes associate with specific chromatin nucleoprotein complexes, regardless of their pattern of DNA integration into the human genome. At least two of the HIV-1 pol-containing nucleoprotein complexes of apparent M(r) approximately 94,000, pI approximately 6.5, and M(r) approximately 47,000, pI approximately 5.1 contain DNA endonuclease activity. This was confirmed in the present study, using linearized pUC19 plasmid substrate. The technique can be used to study a variety of problems concerning the association of specific genes and enzymes with specific nucleoprotein complexes J. Cell. Biochem. Suppls. 32/33:158-165, 1999.

Cell Fractionation↗
PubMed1999

Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites.

The mouse thymidylate synthase (TS) promoter is a member of a family of promoters that lack a TATA box as well as an initiator element and that initiate transcription at many sites over a broad initiation window. An element (MED-1) downstream of the initiation window of almost all promoters of this family has been proposed to be important for promoter activity, as well as for multiple start site utilization. Two consensus MED-1 elements are located downstream of the initiation window of the TS promoter. To determine the role of the MED-1 elements in the TS promoter, one or both elements were inactivated by site-directed mutagenesis and the effects on promoter function were determined. We found that inactivation of the MED-1 elements had no measurable effect on promoter strength, the boundaries of the initiation window, or the pattern of transcriptional start sites. Furthermore, inactivation of the elements did not affect the ability of the TS promoter to direct S phase-specific expression of the gene in growth-stimulated cells. We conclude that the MED-1 element does not play a significant role in TS promoter function and therefore is not an essential component of all TATA-less promoters with complex transcriptional initiation patterns.

Animals↗
PubMed1997

Comet assay in human biomonitoring studies: reliability, validation, and applications.

The comet assay (single-cell gel electrophoresis), which measures DNA strand breaks at the level of single cells, is very easily applied to human lymphocytes, and therefore lends itself to human biomonitoring studies. For the examination of DNA base oxidation (a specific marker of oxidative damage), the assay is modified by including a stage at which the DNA is incubated with a suitable lesion-specific endonuclease. Here we report on the reliability and reproducibility of this approach, from the level of comparing results from duplicate gels prepared from the same sample of cells, up to an assessment of the natural intra- and interindividual variability in lymphocyte DNA damage measured in groups of normal, healthy human volunteers. We applied the assay in investigations of human disease and occupational exposure of factory workers.

Analysis of Variance↗
PubMed1996

DNA base damage induced by ionizing radiation recognized by Escherichia coli UvrABC nuclease but not Nth or Fpg proteins.

Ionizing radiation and other free radical-generating systems induce a great variety of oxidative damage to DNA bases. The major known lesions are repaired by two well-characterized DNA glycosylases of Escherichia coli, endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), which have associated AP lyase activities. To detect and characterize potentially harmful oxidative base DNA lesions that may be repaired by alternative means, we exposed plasmid DNA to low doses of gamma-rays and removed the major base lesions by treatment with Nth and Fpg proteins. The closed circular DNA remaining after these treatments was used as a substrate of the UvrABC endonuclease complex from E. coli and as a template in a DNA polymerase arrest assay in vitro. The circular DNA contained lesions that were recognized and incised by the UvrABC nuclease and also lesions that blocked DNA polymerization in vitro. The blocking lesions were more abundant in DNA irradiated under nitrogen than under air and occurred mainly at tandem guanines; however, they were also frequent at tandem adenines and tandem cytosines.

Bacterial Proteins↗
PubMed1999

Enzymatic and chemical cleavage methods.

Cleavage-based methods of mutation detection offer a simple and intuitive means to detect and in most cases locate mutations within DNA fragment sizes ranging from 500 to 1500 bases. Their main advantages as a presequencing screening technology when scanning for unknown mutations is the potential to increase throughput by multiplexing. Combined with lower reagent costs per sample, mutation scanning methods offer significant advantages over currently available sequencing techniques and are likely to be of increasing importance as genomic sequence data becomes more readily available. Although enzymatic methods offer the advantages of simpler and less hazardous protocols, at present the most robust cleavage methods are based around chemical methods.

Endodeoxyribonucleases↗
PubMed1999

Comparison of detection platforms and post-polymerase chain reaction DNA purification methods for use in conjunction with Cleavase fragment length polymorphism analysis.

The removal of impurities and contaminants from PCR-amplified fragments is important for mutation detection methods which identify mutations based on shifts in electrophoretic mobility. This is particularly critical for assays and detection methods which use target DNA that is labeled prior to analysis and electrophoretic detection. We examined several procedures for purifying DNA amplified by the polymerase chain reaction (PCR) and their use in conjunction with a novel DNA scanning method, the Cleavase fragment length polymorphism (CFLP)* assay. In this study, a 480 bp DNA fragment, fluorescently labeled on the 5'-end of one strand, was amplified and subjected to various widely used purification procedures, including several commercially available clean-up kits. We demonstrate that visualization of the fluorescent label, as opposed to simple ethidium bromide staining, reveals the presence of considerable levels of labeled, truncated, amplification products. The various procedures were evaluated on the basis of their ability to remove these unwanted DNA fragments as well as on the degree to which they inhibited or promoted the CFLP reaction. Several procedures are recommended for use with CFLP analysis, including isopropanol precipitation, gel excision, and several commercially available spin columns. Concurrently, we evaluated (compared) a number of commonly used visualization platforms, including fluorescence imaging, chemiluminescence, and post-electrophoretic staining, for the ability to detect CFLP pattern changes. The advantages and disadvantages of different methods are discussed and amounts of DNA to be used for CFLP analysis on different detection platforms are recommended.

DNA↗
PubMed1999

Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.

BRCA1 Protein↗
PubMed1999

Mutation detection using fluorescent enzyme mismatch cleavage with T4 endonuclease VII.

Mutation detection techniques are often limited by sensitivity, ease of use and short fragment lengths. Enzyme mismatch cleavage (EMC) is a technique capable of rapidly scanning 1 kbp fragments of DNA for mutations. It relies on the ability of a bacteriophage resolvase enzyme, T4 endonuclease VII, to cleave DNA at single base pair mismatches and small heteroduplex loops. Originally the process was performed using radioactively labeled DNA and the results analysed after denaturing polyacrylamide gel electrophoresis and autoradiography. However, access to systems capable of detecting fluorescent species migrating through a gel and the widespread availability of fluorescently tagged primers have greatly improved upon the original technique. A number of mutations were detected using fluorescent EMC and the results compared to performing the technique using radiolabeled DNA. Fluorescent EMC detected the presence, position and number of mutations in DNA fragments as large as 1 kbp. The fluorescent method was found to have advantages over the original method in its ease of use, increase in signal-to-noise ratio and the ability to multiplex samples by labeling DNA fragments with different fluorophores. This improvement on an already established method provides a sensitive, robust technique for mutation detection.

Animals↗
PubMed2000

Single nucleotide polymorphism analyses of the human proliferating cell nuclear antigen (pCNA) and flap endonuclease (FEN1) genes.

The products of proliferating cell nuclear antigen (PCNA) and flap endonuclease (FEN1) genes are multifunctional proteins that are involved in DNA replication and damage repair. Yeast models suggest association of mutant forms of PCNA and FEN1 with genomic instability. In our study, we have determined the single nucleotide polymorphisms in human PCNA and FEN1 genes. We sequenced the coding region and adjacent noncoding region of both the PCNA and FEN1 genes in 120 alleles (60 individuals). In the PCNA gene, we detected 9 sequence variants with Hardy-Weinberg allele frequency ranging from 0.008 to 0.088. No polymorphism was detected in the FEN1 gene. The sequence variants in the PCNA gene included 7 intronic single nucleotide polymorphisms (SNP) and 2 synonymous exonic SNPs. All the intronic SNPs were located in introns 1 and 4, which contain several regulatory elements involved in the control of PCNA gene expression. Six of the 7 intronic SNPs showed complete linkage disequilibrium. We confirmed this allelic linkage disequilibrium by allele-specific PCR sequencing. We genotyped 117 additional individuals belonging to 3 population subgroups using the PCR-RFLP method. Finally, to see if the detected polymorphisms are associated with any cancer type, we genotyped cases with melanomas (37 cases), breast cancers (118 cases) and lung cancers (100 cases). We did not find statistical difference in frequency of polymorphism in any cancer type compared with healthy controls, although in breast cancer the frequency was low. Our results suggest that the coding regions of the PCNA and FEN1 genes are highly conserved when compared with other DNA repair genes. The potential of some of the detected intronic polymorphisms to effect regulation of the PCNA gene expression remains to be determined.

Alleles↗
PubMed2000

Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain.

The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening. MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine. Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1. Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate. The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step. Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites. This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1.

Base Pair Mismatch↗
PubMed2000

Enzymatic mutation detection (EMD) of novel mutations (R565X and R1523X) in the FBN1 gene of patients with Marfan syndrome using T4 endonuclease VII.

The Enzymatic Mutation Detection (EMDtrade mark) method is a streamlined and improved version of the original Enzymatic Cleavage of Mismatch (EMC) method. EMD is a fully homogeneous, rapid four step procedure that allows for detection and localization of mismatched or unmatched nucleotides within heteroduplex DNA. To test the utility of EMD for use in the screening of large and complex genes, the fibrillin 1 (FBN1) gene was scanned in a cohort of six patients diagnosed with connective tissue disorders. Four of the six patients were diagnosed with classic Marfan syndrome (MFS). The results were compared with a previous MDEtrade mark scanning of the same patient cohort. Two causative mutations, R565X and R1523X, were detected by EMD that were not detected by MDE. In both cases, the mutation resulted in premature termination of translation. In addition, several polymorphisms were detected by the enzymatic approach that failed detection by heteroduplex analysis. We propose that the EMD method is a sensitive and rapid approach to mutation detection in large genes such as FBN1.

Adult↗
PubMed2002

Mice deficient for the type II topoisomerase-like DNA transesterase Spo11 show normal immunoglobulin somatic hypermutation and class switching.

Somatic hypermutation in B cells undergoing T cell dependent immune responses generates high affinity antibodies that provide protective immunity. B cells also switch from the expression of immunoglobulin (Ig) M and IgD to that of other Ig classes through somatic DNA recombination. Recent work has implicated DNA strand breaks, possibly DNA double strand breaks (DSB), as the initiating lesions in both class switch recombination and hypermutation, although the etiology of these lesions is not understood. Spo11, a protein structurally related to archaeal type II topoisomerases, generates DSB that initiate meiotic recombination. This characteristic, together with its expression pattern, marks this enzyme as a potential candidate for the initiation of hypermutation, and perhaps also for Ig class switching. To investigate whether Spo11 is involved in these processes, we studied the T cell dependent immune response of Spo11-deficient (Spo11(-/-)) mice against the hapten nitrophenyl (NP). We found that V186.2-bearing IgG1 transcripts had normal levels and patterns of somatic hypermutation. Furthermore, Spo11(-/-) mice showed normal serum levels of all Ig isotypes. These results indicate that Spo11 is not required for Ig hypermutation or class switch recombination.

Animals↗
PubMed1996

Amino acid sequence of peptidyl-prolyl isomerase a of Fusarium sporotrichioides.

Peptidyl-prolyl-cis-trans isomerase catalyzes the interconversion of the cis and trans isomers of the proline-containing polypeptides and the folding process of proteins. This protein was known to be cyclophilin which has high binding affinity for cyclosporin A, a cyclic undecapeptide of fungal origin with potent immunosuppressive property agent. The two cytosolic peptidyl-prolyl-isomerases were found from Fusarium sporotrichioides. The amino acid sequence of the major peptidyl-prolyl isomerase a was determined by conventional sequencing methods; the protein with a calculated molecular mass of 19.7 kDa consisting of 179 amino acids. The comparison of the amino acid sequence of peptidyl-prolyl-isomerase from Fusarium with that of Nucerospora crassa revealed a significant degree of amino acid sequence homology (82.2%).

Amino Acid Isomerases↗
PubMed2003

Phylogenomics of type II DNA topoisomerases.

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.

Animals↗
PubMed1993

RuvA, RuvB and RuvC proteins: cleaning-up after recombinational repairs in E. coli.

After the completion of RecA protein-mediated recombinational repair of daughter-strand gaps in E. coli, participating chromosomes are held together by Holliday junctions. Until recently, it was not known how the cell disengages the connected chromosomes. Accumulating genetic data suggested that the product of the ruv locus participates in recombinational repair and acts after the formation of Holliday junctions. Molecular characterization of the locus revealed that there are three genes--ruvA, ruvB and ruvC; mutations in any one of the genes confer the same phenotype. Recently, the RuvC protein was found to be a Holliday junction resolvase. At first glance, the resolving activity of RuvC alone would appear to be sufficient for the separation of recombining chromosomes. However, in vitro studies show that the filament of RecA protein is unable to dissociate from the products of the recombination reaction. Thus, in vivo, even if the Holliday junctions are resolved by RuvC, RecA filament must be holding two DNA duplexes together. New findings about enzymatic activities of RuvA and RuvB proteins foster the hope that the machinery for removing the RecA filament from DNA has been found.

Bacterial Proteins↗
PubMed1995

DNA packaging and cutting by phage terminases: control in phage T4 by a synaptic mechanism.

Phage DNA packaging occurs by DNA translocation into a prohead. Terminases are enzymes which initiate DNA packaging by cutting the DNA concatemer, and they are closely fitted structurally to the portal vertex of the prohead to form a 'packasome'. Analysis among a number of phages supports an active role of the terminases in coupling ATP hydrolysis to DNA translocation through the portal. In phage T4 the small terminase subunit promotes a sequence-specific terminase gene amplification within the chromosome. This link between recombination and packaging suggests a DNA synapsis mechanism by the terminase to control packaging initiation, formally homologous to eukaryotic chromosome segregation.

Bacteriophage T4↗