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At least 1,711 records · Page 95Linked to original sources
PubMed2006

Fragmented hyaluronan induces transcriptional up-regulation of the multidrug resistance-1 gene in CD4+ T cells.

P-glycoprotein, encoded by the multidrug resistance (MDR)-1 gene, expels various drugs from cells resulting in multidrug resistance. We found previously that interleukin-2, a lymphocyte-activation cytokine, induces P-glycoprotein expression on lymphocytes. Lymphocyte activation involves adhesion with the extracellular matrix, such as hyaluronan, through adhesion molecules on lymphocytes. We investigated the transcriptional regulation of MDR-1 in lymphocytes by fragmented hyaluronan. Fragmented hyaluronan (especially the 6.9-kDa form), not native high molecular hyaluronan, induced translocation of YB-1, a specific transcriptional factor for MDR-1, from the cytoplasm into the nucleus and resulted in the transcription of MDR-1 and the expression of P-glycoprotein on lymphocytes in a dose-dependent manner. Transfection of YB-1 antisense oligonucleotides inhibited P-glycoprotein expression induced by fragmented hyaluronan. The fragmented hyaluronan induced significant P-glycoprotein expression on only activated CD4+ T cells, which highly expressed CD69, and resulted in excretion of intracellular dexamethasone added in vitro. Cyclosporin A, a competitive P-glycoprotein inhibitor, restored intracellular dexamethasone levels in CD4+ T cells. Anti-CD44 monoclonal antibody (Hermes-1) inhibited fragmented hyaluronan-induced YB-1 activation and P-glycoprotein expression in CD4+ T cells. We provide the first evidence that binding of fragmented hyaluronan to CD44 induces YB-1 activation followed by P-glycoprotein expression in accordance with activation of CD4+ T cells. Our findings imply that CD4+ T cell activation by fragmented hyaluronan, induced by characteristic extracellular matrix changes in inflammation, tumors, and other conditions, results in P-glycoprotein-mediated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Accumulation of cAMP in gamma-irradiated thymocytes and internucleosomal DNA fragmentation.

Ionizing radiation, glucocorticosteroids and chemical inducers of differentiation (CID) are cytotoxic to thymocytes, and induce internucleosomal DNA fragmentation. Tissue cAMP levels in thymi of irradiated mice were significantly elevated as early as 30 min post-irradiation. In contrast, cAMP content in the liver was not changed significantly up to 1 h post-irradiation, and then some decrease occurred. Irradiation of isolated thymocytes gave essentially the same results as after irradiation of animals, and the elevation in cAMP 30 min after the irradiation, DNA fragmentation and cell death were linearly related to the dose up to 2.5 Gy. The maximal induction of cAMP level occurs in the fractions of radiosensitive cortical thymocytes. In thymocytes all CID tested also induced the increase in cAMP level with concomitant DNA fragmentation. Unlike ionizing radiation, UVC light did not induce cAMP accumulation and DNA fragmentation in thymocytes. Treatment of UV-irradiated cells with But2 cAMP did not result in an increase in DNA fragmentation. Ionizing radiation induced DNA fragmentation and cell death can be prevented by adding the protein kinases inhibitor H-7. Theophylline was shown to reduce the cAMP response, DNA fragmentation and cell death in gamma-irradiated thymocytes, suggesting that the accumulation of cAMP may be partly related to adenosine receptor sites.

Animals↗
PubMed2004

Oligonucleosome DNA fragmentation of caspase 3 deficient MCF-7 cells in palmitate-induced apoptosis.

Oligonucleosomal fragmentation of nuclear DNA is the late stage hallmark of the apoptotic process. In mammalian apoptotic cells fragmentation is catalyzed by DFF40/ CAD DNase. DFF40/CAD primary activated through site-specific proteolytic cleavage by caspase 3. The absence of caspase 3 in MCF-7 leads to lack of oligonucleosomal DNA fragmentation under numerous apoptotic stimuli. In this study it was shown that palmitate induces apoptotic changes of nuclei and oligonucleosomal DNA fragmentation in casp3 deficient MCF-7. Activation and accumulation of 40-50 kDa DFF40 like DNases in nuclei and cytoplasm of palmitate-treated MCF-7 were detected by SDS-DNA-PAGE assay. Microsomes of apoptotic MCF-7 activate 40-50 kDa nucleases when incubated with human placental chromatin and induce oligonucleosomal fragmentation of chromatin in cell free system. Both DNases activation and chromatin fragmentation are suppressed in presence of caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome associated caspase 7 is suggested to play the principal role in induction of oligonucleosomal DNA fragmentation of casp3 defitient MCF-7.

Animals↗
PubMed1992

Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation.

Our previous report (Muir, D., S. Varon, and M. Manthorpe. 1990. J. Cell Biol. 109:2663-2672) described the isolation and partial characterization of a 55-kD antiproliferative protein found in Schwann cell (SC) and schwannoma cell line-conditioned media and we concluded that SC proliferation is under negative autocrine control. In the present study the 55-kD protein was found to possess metalloprotease activity and stromelysin immunoreactivity. The SC-derived metalloprotease shares many properties with stromelysin isolated from other sources including the ability to cleave fibronectin (FN). Furthermore, limited proteolysis of FN by the SC-derived protease generated a FN fragment which itself expresses a potent antiproliferative activity for SCs. The active FN fragment corresponds to the 29-kD amino-terminal region of the FN molecule which was also identified as an active component in SC CM. Additional evidence that a proteolytic fragment of FN can possess antiproliferative activity for SCs was provided by the finding that plasmin can generate an amino-terminal FN fragment which mimicked the activity of the SC metalloprotease-generated antiproliferative FN fragment. Both the 55-kD SC metalloprotease and the 29-kD FN fragment could completely and reversibly inhibit proliferation of SCs treated with various mitogens and both were largely ineffective at inhibiting proliferation by immortalized or transformed SC lines. Normal and transformed SC types do secrete the proform of stromelysin, however, transformed cultures do not produce activated stromelysin and thus cannot generate the antiproliferative fragment of FN. These results suggest that, once activated, a SC-derived protease similar to stromelysin cleaves FN and generates an antiproliferative activity which can maintain normal SC quiescence in vitro.

Animals↗
PubMed1996

TrkA receptor ectodomain cleavage generates a tyrosine-phosphorylated cell-associated fragment.

The extracellular domain of several membrane-anchored proteins can be released as a soluble fragment by the action of a cell surface endoproteolytic system. This cleavage results in the generation of a soluble and a cell-bound fragment. In the case of proteins with signaling capability, such as tyrosine kinase receptors, the cleavage process may have an effect on the kinase activity of the cell-bound receptor fragment. By using several cell lines that express the TrkA neurotrophin receptor, we show that this receptor tyrosine kinase is cleaved by a proteolytic system that mimics the one that acts at the cell surface. TrkA cleavage is regulated by protein kinase C and several receptor agonists (including the TrkA ligand NGF), occurs at the ectodomain in a membrane-proximal region, and is independent of lysosomal function. TrkA cleavage results in the generation of a cell-associated fragment that is phosphorylated on tyrosine residues. Tyrosine phosphorylation of this fragment is not detected in TrkA mutants devoid of kinase activity, suggesting that phosphorylation requires an intact TrkA kinase domain, and is not due to activation of an intermediate intracellular tyrosine kinase. The increased phosphotyrosine content of the cell-bound fragment may thus reflect higher catalytic activity of the truncated fragment. We postulate that cleavage of receptor tyrosine kinases by this naturally occurring cellular mechanism may represent an additional mean for the regulation of receptor activity.

Animals↗
PubMed2000

A specific activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis.

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal-regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.

Animals↗
PubMed1980

Fibroblast receptor for lysosomal enzymes mediates pinocytosis of multivalent phosphomannan fragment.

Mild acid hydrolysis of phosphomannan secreted by the yeast hansenula holstii (NRRL Y- 2448) produces two phosphomannyl fragments which differ strikingly in their potency as inhibitors of pinocytosis of human beta-glucuronidase by human fibroblasts. The larger molecular weight polyphosphomonoester fragment is 100,000-fold more potent an inhibitor of enzyme uptake than the smaller penta-mannosyl-monophosphate fragment. Binding to attached fibroblasts at 3 degrees C was much greater with the polyphosphomonoester fragment than with the pentamannosyl-monophosphate. The larger molecular weight fragment was also subject to adsorptive pinocytosis and was taken up by fibroblasts at a rate 30- fold greater than the rate of uptake of pentamannosyl-monophosphate. Evidence that the polyphosphomonoester fragment is taken up by the phosphomannosyl-recognition system that mediates uptake of lysosomal enzymes includes: (a) its pinocytosis is inhibited by the same compounds that competitively inhibit enzyme pinocytosis (mannose-6-phosphate and phosphomannan from saccharomyces cerevisiae mutant mnn-1); (b) alkaline phosphatase treatment greatly reduces its susceptibility to pinocytosis; (c) its pinocytosis is competitively inhibited by high-uptake human beta-glucuronidase; and (d) this inhibition by high-uptake enzyme is dramatically reduced by prior treatment of the enzyme with alkaline phosphatase or endoglycosidase-H. Endoglycosidase-H treatment human beta-glucuronidase dramatically reduced its susceptibility to pinocytosis by fibroblasts. The phosphomannosyl components of high- uptake enzyme released by endoglycosidase-H treatment were much less effective inhibitors of polyphosphomonoester pinocytosis than when present on the phosphomannyl-enzyme. These results suggest that high-uptake acid hydrolases may be polyvalent ligands analogous to the polyphosphomonoester mannan fragment whose pinocytosis depends on interaction of more than one phospho-mannosyl recognition marker with pinocytosis receptors on fibroblasts.

Alkaline Phosphatase↗
PubMed1966

The in vivo formation and fate of antigen-antibody complexes formed by fragments and polypeptide chains of rabbit gamma-G-antibodies.

The in vivo formation and subsequent fate of complexes formed between specific rabbit gammaG-antibody subunits and circulating protein antigens was studied in rabbits and guinea pigs. Subunits obtained from purified antibodies were injected immediately after an injection of antigen, and the elimination from the circulation of either I*-labeled gammaG-subunits or labeled antigen determined. In the absence of antigen, all gammaG-subunits which lack the Fc fragment were rapidly eliminated. In the presence of excess antigen, F (ab')(2), Fab and Fd fragments reacted with antigen and remained in the circulation as complexes which were eliminated at the same rate as the antigen. Fab hybrids containing a specific Fd fragment and a nonspecific L chain similarly reacted with antigen and remained in the circulation complexed to antigen. In contrast, L chains and Fab hybrids containing a specific L chain and a nonspecific Fd fragment did not react with antigen in vivo and were rapidly eliminated in both presence and absence of antigen. H chains remained in the circulation of rabbits in the absence of antigen, however, in the presence of antigen, more H chains which formed complexes with antigen remained in the intravascular space and were rapidly eliminated when the immune elimination of the antigen by the host occurred. Nonspecific H chains were rapidly eliminated from the circulaion of guinea pigs, whereas specific H chains remained in the circulation with the antigen. F (ab')(2) fragments formed complexes with antigen near antibody equivalence and in antibody excess which were rapidly eliminated, however, less effectively than complexes formed near antibody equivalence with intact gammaG. Complexes formed in antibody excess between Fab fragments and H chains remained in the circulation at all concentrations studied and were eliminated at the rate of antigen. The role of the Fc fragment in the immune elimination of antigen-antibody complexes is discussed.

Animals↗
PubMed1969

Ultrastructure of papain and pepsin digestion fragments of human IgM globulins.

The ultrastructure of papain and pepsin-digested products of human IgM globulins has been analyzed. Papain digestion was performed both in the presence and absence of cysteine. The Fcmicro fragment was found to represent the central ring structure in the intact IgM molecule, plus a minor part of the appendages extending from the ring. The Fcmicro ring structure was occasionally seen to be composed of dimers of short rods, probably identical with the endpieces of two micro-chains. Such dimeric structures, released from the intact Fcmicro rings, had a tendency to aggregate sidewise, producing complexes of varying size. The dimensions of the Fcmicro fragments were: outer diameter approximately 85 A, inner diameter about 40 A. The length of the protrusions varied from 20-30 A. The Fabmicro preparations contained long strands of sidewise aggregated, short rod-shaped fragments. No aggregates were seen in the F(ab'')(2)micro preparations. The two Fab''micro units in the dimeric F(ab'')(2)micro fragments were usually parallel to each other. The dimensions of the Fabmicro and F(ab'')(2)micro fragments were 50-80 A x 30 A and 75-80 A x 55 A, respectively. These findings provide morphological evidence that the C-terminal ends of the micro-chains (the Fcmicro fragment) make up the central ring structure in the IgM molecule. They further indicate that the F(ab'')(2)micro fragments constitute about (3/4) of the appendages extending from this ring structure.

Binding Sites↗
PubMed1994

Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation.

Apoptosis is the predominant form of cell death observed in a variety of physiological and pathological conditions such as cancer involution, insect metamorphosis, the development of the immune and nervous systems, and embryogenesis. The typical nuclear changes taking place in apoptotic cells include extensive condensation of chromatin and internucleosomal DNA fragmentation into units of 200 base pairs. However, the mechanisms responsible for both chromatin condensation and DNA fragmentation have yet to be elucidated. In this study, micrococcal nuclease and the divalent cations, Ca2+ and Mg2+, were applied to isolated nuclei in an attempt to reconstitute in vitro the digestion of genomic DNA associated with apoptosis. Micrococcal nuclease was found to induce a typical pattern of DNA fragmentation, but did not give rise to chromatin condensation, whereas Ca2+/Mg2+ induced both chromatin condensation and DNA fragmentation in isolated mouse liver nuclei. When the endonuclease inhibitor ZnCl2 was used, the DNA fragmentation induced by Ca2+/Mg2+ in nuclei could be completely inhibited, but chromatin condensation still occurred. For comparison, intact liver cells were treated with valinomycin, a potassium ionophore, which gave rise to an atypical cell death, with chromatin condensation appearing without DNA fragmentation. Our results suggest that endonuclease activation in apoptosis is neither necessary nor sufficient to induce chromatin condensation, and that DNA fragmentation and chromatin condensation may be triggered through separate pathways during apoptosis.

Animals↗
PubMed1998

Expectation and variance of true and false fragment matches in DNA restriction mapping.

Consider a DNA mapping project in which overlap of clones is inferred from multiple complete restriction enzyme digests. Each enzyme cuts each clone randomly into fragments whose lengths are determined with some error. Clones that share fragments with matching lengths could contain a region of overlap. However, common fragment lengths may be due to random coincidence leading to a false overlap declaration. Although the probability of false fragment matching is small, a mapping project involves a large number of clone comparisons. Consequently, erroneous fragment matches can be a serious problem. We use a geometrical probability approach to develop exact integral formulas and first-order approximations for the expected number and variance of classes of fragment pairs that will be identified falsely as matching. We also find exact formulas for the expected value, and variance of the number of true fragment matches. These formulas are useful in comparing different mapping strategies.

DNA↗
PubMed1986

A method for constructing multiple tandem repeats of specific DNA fragments.

We describe a new method for constructing tandem repeats of DNA fragments that allows one to control the number of tandem copies appearing in a final recombinant DNA clone. The principle of the method is to prepare DNA fragments with predetermined cohesive ends and to ligate them together in such a way that unique multimers are generated. First, the fragment of interest is inserted into a vector with a multisite cloning region. Clones are picked with the fragment in both orientations, and the fragments are excised using different pairs of restriction enzymes. The resulting fragments with preprogrammed cohesive ends are mixed, ligated, and digested with one enzyme whose site appears in the cloning region of the vector. From this material multimeric fragments containing specific ends can be isolated and recloned into appropriate vectors. As an example of the method, we show the construction of clones containing either three or six tandem copies of the origin of DNA replication of the mouse polyoma virus. The prospect of using this technique to construct inverted repeat structures is discussed. As part of this work we have constructed a modified M13mp8 vector which contains a unique Xho I cloning site.

DNA Replication↗
PubMedDate not supplied

Site-directed insertion of long single-stranded DNA fragments into plasmid DNA.

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

Base Sequence↗
PubMed2026

fRagmentomics: an R package for integrating cell-free DNA fragment features with mutational status to support liquid biopsy interpretation.

SUMMARY: Liquid biopsy offers a non-invasive approach to study tumor-derived genetic material circulating in plasma. Beyond genetic alterations, the fragmentomic features of cell-free DNA-such as fragment size, genomic position, and end-motifs-provide valuable insights into the biological and clinical context of DNA release. fRagmentomics is a user-friendly R package designed to characterize cfDNA fragments overlapping one or multiple small mutations of any type, starting from an aligned sequencing file (BAM). It supports multiple mutation input formats, accommodates one-based and zero-based genomic conventions, resolves mutation representation ambiguities, and accepts any reference file in FASTA format. For each fragment overlapping a mutation of interest, fRagmentomics outputs fragment-level features including its fragment size, end-motifs, and mutational status, along with additional fragment-level or read-level information. The package implements an indel-aware and optionally soft-clip-preserving fragment size computation that improves accuracy over conventional size estimates based solely on aligned positions. AVAILABILITY AND IMPLEMENTATION: fRagmentomics is licensed under GNU General Public License v3.0 and available at https://github.com/ElsaB-Lab/fRagmentomics, https://anaconda.org/elsab-lab/r-fragmentomics and https://bioconductor.org/packages/fRagmentomics, with documentation and a tutorial. CONTACT: yoann.pradat@gustaveroussy.fr, elsa.bernard@gustaveroussy.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Software↗
PubMed1996

Nucleotide level detection of cyclobutane pyrimidine dimers using oligonucleotides and magnetic beads to facilitate labelling of DNA fragments incised at the dimers and chemical sequencing reference ladders.

We present a method for detecting cyclobutane pyrimidine dimers (CPDs) at the nucleotide level and an adaptation of Maxam-Gilbert sequencing for generating sequence reference ladders. UV irradiated genomic DNA from Escherichia coli was digested with restriction enzyme(s) and incised at the CPDs with Micrococcus luteus UV endonuclease. The subsequent specific fragments were separated using a biotin labelled oligonucleotide containing a sequence complementary to the fragments of interest and streptavidin magnetic beads. These fragments were then radiolabelled on the beads just prior to the running of the sequencing gel. For generating sequence reference ladders, the unlabelled DNA fragments of interest were base-specifically modified and subsequently cleaved at the A+G or C+T sites using the rapid Maxam-Gilbert sequencing treatments. These chemically cleaved fragments can be stored almost indefinitely. Whenever the sequence reference ladders are required, the chemically cleaved fragments can be labelled alongside the CPD-specifically incised DNA fragments using the same procedure. The adaptation of the method to detect other types of DNA damage is also discussed.

Base Sequence↗
PubMed1999

Urinary trypsin inhibitor down-regulates hyaluronic acid fragment-induced prostanoid release in cultured human amnion cells by inhibiting cyclo-oxygenase-2 expression.

We postulated that urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, may inhibit low molecular weight hyaluronic acid (HA) fragment-induced prostanoid release and de-novo expression of the inducible cyclo-oxygenase-2 (COX-2) isoform in human term amnion cells. Purified amnion cultures were obtained from human fetal membranes and were exposed to a HA fragment (molecular weight 35 kDa) in the presence or absence of UTI (0-5.0 micromol/l). Amnion cells treated with the HA fragment (100 nmol/l) released significantly more prostanoids (PGE2 and PGF2alpha) than controls (PGE2: 2.1 +/- 0.13 pg/10(6) cells/24 h compared with 0.42 +/- 0.01, P < 0.05; PGF2alpha: 1.0 +/- 0.17 pg/10(6) cells/24 h compared with 0.13 +/- 0.01, P < 0.05). UTI inhibited HA fragment-induced prostanoid release in a dose-dependent manner, with 50% inhibitory concentration values of 0.8 micromol/l for PGE2 and 1.9 micromol/l for PGF2alpha. Western blot analyses demonstrated that protein levels of COX-2 were substantially increased in amnion cells treated with HA fragment. HA fragment-mediated COX-2 production was markedly diminished by pretreatment with UTI (1.0 micromol/l). These results are the first to demonstrate that UTI is a potent inhibitor of HA fragment-induced arachidonic acid metabolism.

Amnion↗
PubMed2003

Expression of apoptosis-related genes during human preimplantation embryo development: potential roles for the Harakiri gene product and Caspase-3 in blastomere fragmentation.

In order to resolve the mechanisms and reasons of cellular fragmentation it is crucial to understand what genes may be responsible for regulation of this process. We report herein that human oocytes and preimplantation embryos possess abundant levels of transcripts encoding cell death suppressors, Mcl-1, Bcl-x and Bag-1, and the cell death inducer genes, Bax and Caspase-2. Lower but detectable levels of mRNA expression for the Bfl-1/a1, Bcl-w, Harakiri (Hrk) and Caspase-3 genes were also detected during all developmental stages. We also performed analysis of gene expression in single human embryos exhibiting various degrees of fragmentation at the 2-, 4- and 8-cell stages. At the 4-cell stage, embryos displaying 30-50% fragmentation showed a significant increase in Hrk mRNA levels (P = 0.016). Immunostaining with anti-Hrk antibody confirmed increased staining in some, but not all, fragmented embryos. While Caspase-3 transcripts were elevated in both 4- and 8-cell embryos exhibiting a severe degree of fragmentation, this difference did not reach statistical significance. However, accumulation of Caspase-3 mRNA in fragmented embryos was paralleled by an induction of Caspase-3-like activity. These findings suggest that cellular fragmentation in a subset of human preimplantation embryos could be regulated by certain components of a genetic programme of cell death.

Alternative Splicing↗
PubMed1985

Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis.

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.

Animals↗