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Plasma concentrations and total amounts of pregnancy associated plasma protein A were determined in 272 patients at 34 weeks'' gestation by immunoelectrophoretic assay. The mean plasma concentration and mean total amount of this protein were closely related (r = 0.9643) and were significantly raised in patients who subsequently developed pre-eclampsia (28 patients), went into premature labour (12), or suffered from antepartum haemorrhage (10). Mean values in all patients delivering growth-retarded babies were also raised, but when the results for such patients who also had other complications were excluded there were no differences between the sets of means. The assays were easily performed, and they may be a useful technique for screening pregnant women to detect those at risk of developing pre-eclamptic toxaemia, although the full potential of these assays cannot be realised until the protein''s function is known. 相似文献
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Actinomycin D has long been known to bind selectively to the dinucleotide step GpC. We have investigated its ability to bind to other non-canonical sequences using a series of synthetic DNA fragments. DNase I footprinting experiments reveal that actinomycin can also bind well to GG (CC). Binding to this sequence and the canonical GC site is potentiated by flanking regions of (GT)n.(AC)n. Weaker but specific binding to GT and AC is also evident and appears to be cooperative. 相似文献
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Sequence-specific binding property of Arabidopsis thaliana telomeric DNA binding protein 1 (AtTBP1) 总被引:1,自引:0,他引:1
We have identified an Arabidopsis thaliana cDNA, designated as AtTBP1, encoding a protein with a predicted size of 70.6 kDa that specifically binds to the plant telomeric repeat sequence TTTAGGG. AtTBP1 is present as a single-copy gene in Arabidopsis genome and is expressed ubiquitously in various organs. AtTBP1 has a single Myb telomeric DNA binding domain at the C-terminus and an extensive homology with other known telomere-binding proteins. The isolated C-terminus of AtTBP1 is capable of sequence-specific DNA binding to plant duplex telomeric DNA. These results suggest that AtTBP1 may play important roles in plant telomere function in vivo. 相似文献
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Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites 总被引:48,自引:0,他引:48
The statistics of base-pair usage within known recognition sites for a particular DNA-binding protein can be used to estimate the relative protein binding affinities to these sites, as well as to sites containing any other combinations of base-pairs. As has been described elsewhere, the connection between base-pair statistics and binding free energy is made by an equal probability selection assumption; i.e. that all base-pair sequences that provide appropriate binding strength are equally likely to have been chosen as recognition sites in the course of evolution. This is analogous to a statistical-mechanical system where all configurations with the same energy are equally likely to occur. In this communication, we apply the statistical-mechanical selection theory to analyze the base-pair statistics of the known recognition sequences for the cyclic AMP receptor protein (CRP). The theoretical predictions are found to be in reasonable agreement with binding data for those sequences for which experimental binding information is available, thus lending support to the basic assumptions of the selection theory. On the basis of this agreement, we can predict the affinity for CRP binding to any base-pair sequence, albeit with a large statistical uncertainty. When the known recognition sites for CRP are ranked according to predicted binding affinities, we find that the ranking is consistent with the hypothesis that the level of function of these sites parallels their fractional saturation with CRP-cAMP under in-vivo conditions. When applied to the entire genome, the theory predicts the existence of a large number of randomly occurring "pseudosites" with strong binding affinity for CRP. It appears that most CRP molecules are engaged in non-productive binding at non-specific or pseudospecific sites under in-vivo conditions. In this sense, the specificity of the CRP binding site is very low. Relative specificity requirements for polymerases, repressors and activators are compared in light of the results of this and the first paper in this series. 相似文献
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A DNA sequencing technique was applied to the highly reiterated DNA from HeLa S3 cells in order to detect DNA damage induced by the antitumor drug, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). A DNA reiterated fragment of 92 base pairs (bp) was isolated by gel electrophoresis after EcoRI and EcoRI restriction endonuclease digestion. In the defined sequence of the 92 bp fragment, ACNU caused damage and modifications primarily at guanine moieties, leading to alkali-labile sites as determined by subsequent piperidine reaction on an extended Maxam-Gilbert sequencing gel. These results indicate that guanine moieties in double-stranded DNA are preferentially vulnerable to ACNU over other base moieties. 相似文献
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It has been reported that 14C-labeled methyl-n-amyl ketone (MAK, 2-heptanone) is able to bind spontaneously, in vitro, to isolated rat liver DNA to the extent of 400 pmol/mg DNA; and that 14C-MAK, when given by gavage to female Fischer 344 rats, resulted in HPLC chromatograms of isolated, hydrolyzed liver DNA in which some radiolabel was not associated with the four normal DNA bases dA, dT, dC, and dG. The present studies were undertaken to re-examine the hypothesis that MAK is able to bind to rat liver DNA. In the in vitro study, liver nuclear DNA was incubated with [2-14C]-labeled MAK (25 mCi/mmol) in the absence, or in the presence of rat liver microsomes, precipitated, washed free of unbound MAK, and counted by scintillation spectrometry. No binding to DNA by MAK was detectable. In the in vivo study, groups of five female F344 rats were exposed by inhalation to 0, 80, 400, or 1000 ppm MAK for 6 h/day for 10 days. DNA was purified from the liver nuclei of the 0 and 1000 ppm dosed animals, and 32P-postlabeling techniques were used to assay for adducts. No DNA adducts were detected using these techniques. It was concluded that MAK lacks the ability to bind to rat liver DNA in vitro and in vivo. 相似文献
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The binding of mouse liver chromosomal proteins to DNA has been investigated using the nitrocellulose filter binding technique. Careful purification of the DNA involving nuclease S1 digestion and prefiltration through a nitrocellulose filter is used to reduce background binding in the absence of protein to less than 1%. Procedures involving direct binding of protein to labeled DNA, competition of binding of labeled DNA by unlabeled DNA, and dissociation of DNA . protein complexes with time do not indicate significant preference for binding to mouse DNA relative to Escherichia coli DNA. This specificity is demonstrated much more clearly by a novel type of procedure, which we call a sequential binding procedure. In this procedure non-specific binding proteins are sequestered by incubation with an excess of unlabeled E. coli DNA prior to addition of labeled DNA. Under these conditions, labeled mouse DNA is bound to filters to a 3- to 4-fold greater extent than labeled E. coli DNA. 相似文献
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Chromosomal distribution of heterochromatin protein 1 (HP1) in Drosophila: a cytological map of euchromatic HP1 binding sites 总被引:2,自引:0,他引:2
The Heterochromatin Protein 1 (HP1) is a conserved protein which is best known for its strong association with the heterochromatin of Drosophila melanogaster. We previously demonstrated that another important property of HP1 is its localization to the telomeres of Drosophila, a feature that reflects its critical function as a telomere capping protein. Here we report our analysis of the euchromatic sites to which HP1 localizes. Using an anti-HP1 antibody, we compared immunostaining patterns on polytene chromosomes of the Ore-R wild type laboratory strain and four different natural populations. HP1 was found to accumulate at specific euchromatic sites, with a subset of the sites conserved among strains. These sites do not appear to be defined by an enrichment of known repetitive DNAs. Comparisons of HP1 patterns among several Drosophila species revealed that association with specific euchromatic regions, heterochromatin and telomeres is a conserved characteristic of HP1. Based on these results, we argue that HP1 serves a broader function than typically postulated. In addition to its role in heterochromatin assembly and telomere stability, we propose that HP1 plays an important role in regulating the expression of many different euchromatic regions. 相似文献
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The herpes simplex virus 1 origin binding protein: a DNA helicase. 总被引:31,自引:0,他引:31
R C Bruckner J J Crute M S Dodson I R Lehman 《The Journal of biological chemistry》1991,266(4):2669-2674
A recombinant herpes simplex 1 origin binding protein, the product of the herpes UL9 gene, has been overexpressed in mammalian cells and purified to near homogeneity. The origin binding protein shows DNA-dependent nucleoside 5'-triphosphatase and DNA helicase activities in addition to its origin binding activity. The ability to hydrolyze nucleoside 5'-triphosphates is influenced strongly by the structure and sequence of the DNA cofactor. The properties of the recombinant origin binding protein are identical to those of the protein synthesized in herpes simplex 1-infected mammalian cells. 相似文献
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Isolation and cloning of putative mouse DNA replication initiation sites: binding to nuclear protein factors.
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By using an original two-step technique (trioxsalen crosslinking/immunoprecipitation) we were able to isolate in a single-stranded form a fraction of mouse DNA enriched in putative Replication Initiation Sequences (RIS). The isolated and purified single-strand fragments were made double-stranded in vitro and were cloned in pUC12 to prepare a confined RIS library. 30 randomly selected RIS inserts were subjected to gel mobility shift assay using nuclear extracts either from dividing, or from quiescent mouse cells. Twelve out of the 30 RIS fragments showed specific binding to proteins present in nuclear extract from dividing cells, while none were retarded by extracts from quiescent cells. RIS12, RIS18 and RIS30 were sequenced and it was found that they were A+T rich and contained different regulatory elements. By using a two step procedure (Heparin-sepharose chromatography/DNA affinity chromatography) we isolated the protein factor that specifically binds to RIS12. It appeared as a double band with apparent molecular masses of 63 and 65 kD. 相似文献
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Several hundred copies of a highly conserved extragenic palindromic sequence, 20-40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid-associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid. 相似文献
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Botros M Hallberg M Johansson T Zhou Q Lindeberg G Frändberg PA Tömböly C Tóth G Le Grevès P Nyberg F 《Peptides》2006,27(4):753-759
Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors. 相似文献
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Characterization of a mitochondrial protein binding to single-stranded DNA. 总被引:7,自引:3,他引:4
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A DNA-binding protein from Xenopus laevis oocyte mitochondria which has been found associated with the D-loop also shows a strong preference for single-stranded DNA. The binding to polynucleotides is dependent on the base composition, but no sequence specificity was found. This protein, called mtSSB, binds tightly and cooperatively to single-stranded DNA. By its amino-acid composition and its binding properties it appears to be similar to the single-stranded DNA-binding proteins found in prokaryotes. 相似文献
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A photochemical method to map ethidium bromide binding sites on DNA: application to a bent DNA fragment 总被引:4,自引:0,他引:4
It is shown that, when irradiated in the visible, ethidium bromide (EB) engages in direct photochemistry with its DNA binding site. At the photochemical end point, an average of one single-strand break is produced per bound EB molecule in a reaction which also bleaches the dye chromophore. Using high-resolution electrophoresis, we have mapped the distribution of EB photocleavage sites on DNA, at one-base resolution. It is argued that because the photocleavage is stoichiometric, the resulting pattern is similar to, if not identical with, the local distribution of EB binding affinity. When interpreted in the context of the extensive thermodynamic and structural data which are available for EB, a binding distribution of that kind can be used to infer details of DNA structure variation within the underlying helix. As a first application of the method, we have used EB to probe the structure of a 265 bp fragment of DNA, which had been described as being bent as the result of a periodic array of oligo(A) segments [Kitchin et al. (1986) J. Biol. Chem. 261, 11302]. The EB mapping data provide evidence that the oligo(A) elements in this fragment assume a local secondary structure which is different than that assumed by isolated ApA nearest neighbors and that the ends of the oligo(A) elements comprise a junctional domain with EB binding properties which differ from those of the oligo(A) element or of random-sequence DNA. 相似文献
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Purification of Rts1 RepA protein and binding of the protein to mini-Rts1 DNA. 总被引:9,自引:7,他引:2
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RepA protein, essential for the replication of plasmid Rts1, was purified, and its binding to mini-Rts1 subregions was examined by a DNase I protection assay. RepA protected the incI and incII iterons, a region immediately upstream of the repA promoter, and a 10-base-pair region located between the most external incII iteron and a GATC box. The protection was less efficient when preheated RepA was used. 相似文献