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1.
S. A. M. Martins D. M. F. Prazeres L. P. Fonseca G. A. Monteiro 《Biotechnology letters》2010,32(2):229-234
Klenow I polymerase activity was combined with solid phase DNA hybridization to detect non-amplified genomic DNA (gDNA) sequences
from Escherichia coli. Aminopropyl-controlled pore glass surface-bound oligonucleotides were hybridized to fragmented gDNA. The template-mediated
extension at the 3′-terminus of the immobilized probe was then promoted in the presence of Klenow I polymerase and digoxigenin-labeled
nucleotides. Detection of the extended probes was accomplished with an anti-digoxigenin alkaline phosphatase conjugate protocol
coupled to colorimetric or fluorescent detection. Using the colorimetric protocol, the proof-of-concept was established. The
fluorescence-based methodology, on the other hand, provided the basis for a quantitative interpretation of the data, affording
a detection limit of 5 pM gDNA. 相似文献
2.
Lahja Uitto Jussi Halleeen P?ivi Remes Tapio Kesti Juhani E. Syv?oja 《Chromosoma》1992,102(Z1):S142-S146
The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes
mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide
non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary
regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to
those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA
is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a
non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition
to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase
epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity. 相似文献
3.
Cleavage of double-stranded DNA was performed with cationic manganese porphyrin complexes linked via a spermine tether to
the 3′- or 5′-side of triple-helix-forming oligonucleotides (cleaver-TFO conjugates). The targeted sequence was a 15-polypurine
sequence present in the env gene of HIV-1 (positions 7301–7315). The presently used TFOs contain only thymine and 5-methylcytosine residues and one adenine
at the 3′-end in order to be able to easily introduce a 3′-polyamine linker by reductive amination of the corresponding 3′-apurinic
polypyrimidine oligonucleotides. With this method we prepared these TFO-cleaver conjugates in 45% yield with only two equivalents
of the Mn-TrisMPyP-COOH precursor. These new metalloporphyrin-TFO conjugates were able to cleave a complementary 45-mer duplex
at 10 nM concentration with only ten equivalents of TFO-cleaver. Conjugates without spermine, without 5-methylcytosine, with
a random sequence or with the managanese porphyrin-spermine entity on the 5′-end of TFOs were synthesized for comparative
studies.
Received: 6 December 1995 / Accepted: 5 February 1996 相似文献
4.
In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle
bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively
low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation
indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression.
Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997 相似文献
5.
Proteins diffusing from tobacco pollen grains exhibit different phosphohydrolytic activities. Molecular sieving produces nuclease
fractionation into forms I, II and III with apparent molecular masses ≥ 60 × 103, 32.9 × 103 and 24.6 × 103, respectively, and separation of principal forms II and III from phosphatase and major part of 5′- and 3′-nucleotidase activities.
These forms did not differ in the mode of substrate attack and were combined for further enzyme characterization.
The preparation had 3′-nueleotidase activity even after further purification by DEAE-cellulose chromatography. The enzyme
is an endonuclease with preference for single stranded molecules. The endolytical cleavage of native DNA occurs simultaneously
in both strands and generates limit products of about 58 pairs of nucleotides. DNA duplex polymers are also cleaved by a terminally-directed,
exonuclease-like process. The products of DNA degradation are oligonucleotides and 5′-mononucleotides. In the presence of
NaCl, both endolytical and exonucleaselike activities on bihelical DNA are inhibited and the proportion of mono-to oligonucleotides
produced increases. The enzyme can rapidly convert superhelical plasmid DNA to a nicked open circular form, and then to a
unit-length linear molecule.
On the basis of these properties and of those found earlier (sugar-unspecificity, acidic pH optimum, activation by Zn2+ ions), the extracellular nuclease of tobacco pollen can be classified as plant nuclease I (EC 3.1.30.x). 相似文献
6.
L. M. Skobeltsyna D. V. Pyshnyi I. G. Shishkina D. R. Tabatadze G. M. Dymshits V. F. Zarytova E. M. Ivanova 《Molecular Biology》2000,34(3):321-327
A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic
ligation of a tandem of three short oligonucleotides B∼pN8+pN4+pN′8 Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN8 is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′8 Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide
on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and
chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide)
is as low as ∼10−14 mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA
template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads.
This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified
DNA fragments. 相似文献
7.
Summary. Phosphate transport in bacteria occurs via a phosphate specific transporter system (PSTS) that belongs to the ABC family of
transporters, a multisubunit system, containing an alkaline phosphatase. DING proteins were characterized due to the N-terminal
amino acid sequence DINGG GATL, which is highly conserved in animal and plant isolates, but more variable in microbes. Most
prokaryotic homologues of the DING proteins often have some structural homology to phosphatases or periplasmic phosphate-binding
proteins. In E. coli, the product of the inducible gene DinG, possesses ATP hydrolyzing helicase enzymic activity. An alkaline phosphorolytic enzyme of the PSTS system was purified
to homogeneity from the thermophilic bacterium Thermus thermophilus. N-terminal sequence analysis of this protein revealed the same high degree of similarity to DING proteins especially to
the human synovial stimulatory protein P205, the steroidogenesis-inducing protein and to the phosphate ABC transporter, periplasmic
phosphate-binding protein, putative (P. fluorescens Pf-5). The enzyme had a molecular mass of 40 kDa on SDS/PAGE, exhibiting optimal phosphatase activity at pH 12.3 and 70 °C.
The enzyme possessed characteristics of a DING protein, such as ATPase, ds endonuclease and 3′ phosphodiesterase (3′-exonuclease)
activities and binding to linear dsDNA, displaying helicase activity on supercoiled DNA. Purification and biochemical characterization
of a T. thermophilus DING protein was achieved. The biochemical properties, N-terminal sequence similarities of this protein implied that the
enzyme belongs to the PSTS family and might be involved in the DNA repair mechanism of this microorganism.
Authors’ address: Assist. Prof. A. A. Pantazaki, Laboratory of Biochemistry, Department of Chemistry, Aristotle University
of Thessaloniki, Thessaloniki 54124, Greece 相似文献
8.
A. M. Varizhuk S. V. Kochetkova N. A. Kolganova E. N. Timofeev V. L. Florent’ev 《Russian Journal of Bioorganic Chemistry》2010,36(2):199-206
A dinucleotide containing a C3′-NH-C(O)-CH2-C5′ amide internucleotide bond was synthesized by the interaction of 3′-deoxy-3′-amino-5′-O-(tert-butyldimethylsilyl)thymidine with 3′-O-benzyl-2′-O-tert-butyldimethylsilyl-5′-deoxy-5′-carboxymethylribosylthymine, which was obtained from 2′-O-acetyl-3′-O-benzyl-5′-deoxy-5′-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation
of liberated hydroxyl and ester saponification. After standard manipulation with protecting groups, the dinucleotide was converted
into 3′-O(2-cyanoethyl-N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The
melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting
temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond
into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified
ones. 相似文献
9.
10.
Herschkovitz Y Lerner A Davidov Y Rothballer M Hartmann A Okon Y Jurkevitch E 《Microbial ecology》2005,50(2):277-288
Inoculation with Azospirillum brasilense exerts beneficial effects on plant growth and crop yields. In this study, a comparative analysis of maize (Zea mays) root inoculated or not inoculated with A. brasilense strains was performed in two soils. Colonization dynamics of the rhizobacteria were tracked in various root compartments
using 16S rRNA-targeted probes and 4′,6′diamidino-2-phenylindole staining, and the structure of bacterial populations in the
same samples was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction products of the 16S
rRNA gene. Based on whole cell hybridization, a large fraction of the bacterial community was found to be active in both the
rhizoplane–endorhizosphere and rhizosphere soil compartments, in both soil types. A DGGE fingerprint analysis revealed that
plant inoculation with A. brasilense had no effect on the structural composition of the bacterial communities, which were also found to be very similar at the
root tip and at zones of root branching. However, rhizobacterial populations were strongly influenced by plant age, and their
complexity decreased in the rhizoplane–endorhizosphere in comparison to rhizosphere soil. A clone library generated from rhizosphere
DNA revealed a highly diverse community of soil and rhizosphere bacteria, including an indigenous Azospirillum-like organism. A large proportion of these clones was only distantly related to known species.
Herschkovitz and Lerner contributed equally to this work. 相似文献
11.
Summary 5′-Nucleotidase and alkaline phosphatase activity was investigated in the developing kidney of the mouse by histochemical
and electrophoretic methods. The growth of the kidneys was studied by determining the incorporation of radioactive thymidine
by autoradiography. During development the isoenzyme patterns of 5′-nucleotidase and alkaline phosphatase behaved in a different
way. In correlating the histochemical and electrophoretic changes, it has been found that the 5′-nucleotidase isoenzymes as
well as the alkaline phosphatase isoenzymes are located in different parts of the kidney. In the convoluted part of the proximal
tubule 5′-nucleotidase isoenzyme 3 and alkaline phosphatase isoenzyme 5 are present, while in the straight part of this tubule
5′-nucleotidase isoenzyme 5 and — upto three weeks — alkaline phosphatase isoenzyme 3 are located. So in tissue structures
having different functional capacities, different isoenzymes of 5′-nucleotidase and alkaline phosphatase are found. 相似文献
12.
Toshihiro Kobayashi Vadim S. Zinchuk Teruhiko Okada Eva Garcia del Saz H. Seguchi 《Histochemistry and cell biology》1998,110(4):395-406
Human neutrophils possess alkaline phosphatase-containing intracellular granules which are upregulated to the cell surface
upon stimulation. The mechanism that governs the intracellular dynamics of these granules is, however, poorly understood.
The aim of the present study was to investigate the possible participation of GTP-binding proteins in the reorganization and
exocytosis of the alkaline phosphatase-containing granules using electropermeabilized cells. Biochemical assays using intact
neutrophils showed that the alkaline phosphatase activity was upregulated and exocytosed into the extracellular space upon
stimulation with AlF4
– and N-formyl peptide. This upregulation was inhibited by treatment of cells with pertussis toxin and botulinum toxin. Alkaline
phosphatase activity was also upregulated in electropermeabilized cells stimulated with guanosine 5′-O-(3-thiotriphosphate) (GTPγS), but not with guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Cytochemically, alkaline phosphatase-containing granules were dispersed throughout the cytoplasm
in unstimulated electropermeabilized neutrophils. Upon stimulation with GTPγS, but not with GDPβS, these granules fused to
form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorganization
of the alkaline phosphatase-containing granules in cells stimulated with GTPγS. The results from this study indicate that
GTP-binding proteins participate in the reorganization and exocytosis of alkaline phosphatase-containing granules associated
with the microtubules in electropermeabilized human neutrophils.
Accepted: 31 March 1998 相似文献
13.
Kohji Hasunuma 《Journal of plant research》1985,98(3):203-217
A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase,
EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not
only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them.
In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only
limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase
activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM. 相似文献
14.
The structural gene for DNA polymerase I of Rhizobium leguminosarum was determined. The rhizobium DNA polymerase I consists of 1016 amino acid residues with a calculated molecular weight of
111,491 Dalton. The amino acid sequence comparison with E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, and Rickettsia prowazekii DNA polymerase I showed that, although 5′-nuclease and DNA polymerase domains are highly conserved, 3′ to 5′ exonuclease
domains are much less conserved. While both R. leguminosarum and R. prowazekii belong to the alpha subdivision of the Proteobacteria on the basis of 16S ribosomal RNA phylogeny, the primary structure of the DNA polymerase I is quite different; the rhizobium
DNA polymerase I has 3′ to 5′ proofreading exonuclease, but the rickettsia DNA polymerase I does not.
Received: 15 December 1998 / Accepted: 2 February 1999 相似文献
15.
Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.) 总被引:3,自引:0,他引:3
Nahm SH Lee HJ Lee SW Joo GY Harn CH Yang SG Min BW 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2005,111(6):1191-1200
In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program. 相似文献
16.
Salmonella causes the majority of infections in humans and homeothermic animals. This article describes a specific polymerase chain
reaction (PCR) method developed for a rapid identification of Salmonella. A gyrB-targeted species-specific primer pair, S-P-for (5′-GGT GGT TTC CGT AAA AGT A-3′) and S-P-rev (5′-GAA TCG CCT GGT TCT TGC-3′),
was successfully designed. PCR with all the Salmonella strains produced a 366- bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 0.01 ng with genomic DNA or 3.2 cells per assay. Good specificity was
also demonstrated by fecal samples, from which only the gyrB gene of Salmonella was amplified. Using the culture-PCR method, 27 isolates on Salmonella-Shigella (SS) medium were rapidly identified as Salmonella, which was confirmed by the sequencing of the gyrB gene. 相似文献
17.
L. S. Robert M. Lévesque-Lemay J. L. Gerster H.-P. Hong W. Keller 《Plant cell reports》1999,18(5):357-362
A genomic clone, Pis G363, containing the Brassica napus stigma-expressed gene Pis 63-2 was isolated and sequenced. The coding region of Pis G363 does not possess introns and shows
82% identity to the nucleotide sequence of a gene from Arabidopsis BAC clone T01B08. A 2-kb promoter fragment from Pis G363 was fused to the coding sequence of the marker enzyme β-glucuronidase (GUS) and introduced into tobacco via Agrobacterium-mediated transformation. The promoter fragment directed expression of the GUS gene in the stigma of transgenic tobacco. Some
transformants also showed relatively low GUS activity in the pollen.
Received: 25 May 1998 / Revision received: 30 July 1998 / Accepted: 21 August 1998 相似文献
18.
P. E. Vorobjev I. A. Pyshnaya D. V. Pyshnyi M. N. Repkova A. G. Venyaminova M. A. Zenkova E. M. Ivanova C. Scalfi-Happ H. Seliger G. M. Bonora V. F. Zarytova 《Russian Journal of Bioorganic Chemistry》2000,26(8):758-764
The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol
linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number
of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for theE. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2–1.4-fold.
It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers.
RNase H simultaneously hydrolyzed the RNA 3′-ends of each hybrid duplex involving a bridged oligonucleotide. The presence
of an inverted 3′-3′-phosphodiester bond at the 3′-end of the oligodeoxyribonucleotide only slightly affected the RNase H
activity.
For the previous report, see [1]. 相似文献
19.
An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible
basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNAAsn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region)
nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive
sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons).
The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on
the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B
DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close
relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe.
As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and
Pol γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol γ from yeast
to human.
Received: 13 October 1998 / Accepted: 21 December 1998 相似文献
20.
Estimates of thylakoid electron transport rates (Je) from chlorophyll fluorometry are often used in combination with leaf gas exchange measurements to provide detailed information
about photosynthetic activity of leaves in situ. Estimating Je requires accurate determination of the quantum efficiency of Photosystem II (ΦP), which in turn requires momentary light saturation of the Photosystem II light harvesting complex to induce the maximum
fluorescence signal (FM′). In practice, full saturation is often difficult to achieve, especially when incident photosynthetic photon flux density
(Q) is high and energy is effectively dissipated by non-photochemical quenching. In the present work, a method for estimating
the true FM′ under high Q was developed, using multiple light pulses of varying intensity (Q′). The form of the expected relationship
between the apparent FM′ and Q′ was derived from theoretical considerations. This allowed the true FM′ at infinite Q′ to be estimated from linear regression. Using a commercially available leaf gas exchange/ chlorophyll fluorescence
measurement system, Je was compared to gross photosynthetic CO2 assimilation (AG) under conditions where the relationship between Je and AG was expected to be linear. Both in C4 leaves (Zea mays) in ambient air and also in C3 leaves (Gossypium hirsutum) under non-photorespiratory conditions the apparent ratio between Je and AG declined at high Q when ΦP was calculated from FM′ measured simply using the highest available saturating pulse intensity. When FM′ was determined using the multiple pulse / linear regression technique, the expected relationship between Je and AG at high Q was restored, indicating that the ΦP estimate was improved. This method of determining FM′ should prove useful for verifying when saturating pulse intensities are sufficient, and for accurately determining ΦP when they are not.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献