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At least 1,639 records · Page 91Linked to original sources
PubMed1994

A short purine oligonucleotide forms a highly stable triple helix with the promoter of the murine c-pim-1 proto-oncogene.

A homopurine-homopyrimidine region of murine c-pim-1 proto-oncogene was chosen as a target for triple-helix-forming oligonucleotide. Oligonucleotide 5'-GGG-GAGGGGGAGG-3' was shown to bind to its target sequence in the presence of 50 mM Na+ or K+, 10 mM MgCl2 and 20 mM Tris-acetate, pH 7.5. This oligonucleotide is bound in an antiparallel orientation with respect to the homopurine sequence. As was shown by co-migration assay the triplex is stable up to 65 degrees C. At 37 degrees C it was practically irreversible: after 24 hours of co-migration assay there was no traces of triplex dissociation. The rate of triplex formation was highly accelerated with increase of temperature and Mg2+ concentration. This rate was higher for superhelical DNA when compared to the linear and circular ones and the preference was dependent from temperature and Mg2+ concentration. The precision of this interaction is extremely high: sequences in c-pim-1 promoter region with only one substitution when compared to the target gave negligible triplex formation under investigated conditions. These data suppose that natural triplex structures could play an important role in eukaryotic gene regulation and/or chromatin structure formation.

Animals↗
PubMed1994

Relative contribution of photo-addition, helper oligonucleotide and RNase H to the antisense effect of psoralen-oligonucleotide conjugates, on in vitro translation of Leishmania mRNAs.

We investigated the properties of two antisense oligonucleotides, 11 alpha Pso and 14TMP, 11 and 14 nucleotides long, respectively, and conjugated to psoralen derivatives. These oligonucleotides were complementary to the mini-exon sequence of Leishmania amazonensis. Upon ultraviolet (UV) irradiation these oligomers were selectively cross-linked to DNA or RNA target sequences, either 14 or 35 nucleotides long. The yield of photo-addition was much lower on the longer targets than on the shorter ones, due to the presence of a hairpin structure. The co-addition of a helper oligonucleotide, whose binding site, on the 35-mer, was adjacent to that of the psoralen-derivatized antisense oligomer, improved the cross-linking efficiency. We then determined the effect of 14TMP on in vitro translation of Leishmania mRNA in cell-free extracts. Non-irradiated antisense oligonucleotide/mRNA complexes reduced the protein synthesis in wheat germ extract but not in rabbit reticulocyte lysate. Conversely, UV irradiation induced a 14TMP-dependent reduction of translation in reticulocyte lysate whereas the inhibition was not improved in the wheat germ extract. These results are discussed with respect to the involvement of RNase-H in the oligonucleotide-mediated effect on protein synthesis.

Animals↗
PubMed1998

DNA ribonucleases that are active against intracellular hepatitis B viral RNA targets.

DNA ribonucleases directed against direct repeat 1 (DR1) and polyadenylation signal regions of hepatitis B virus (HBV) messages were prepared with phosphorothioate modifications and varying arm lengths. DNA ribonucleases modified throughout the entire molecule and in the target binding arms were completely protected from degradation after incubation with serum. DNA ribonuclease modified only at the 5' and 3' termini remained 92.9% intact after incubation. Molecules with no modification were degraded to 67.6% under the same conditions. However, modification of the entire molecule and in the recognition arms resulted in 99.8% and 98.4% inactivation of cleavage activity, respectively. Modification of only the termini resulted in retention of 20% to 40% of original activity. Lengthening each terminally modified arm from 9 to 11 nucleotides increased cleavage efficiency almost 10-fold. In Huh 7 cells, DR1-directed DNA ribonucleases with terminal modifications significantly suppressed HBV-luciferase fusion gene expression up to 48% of control. In contrast, DNA ribonucleases had no effect on a control construct lacking any HBV target sequences. Moreover, inactivated mutant and HCV-directed DNA ribonucleases had no significant effects on the HBV target. We conclude that resistance of DNA ribonucleases to degradation can be enhanced through phosphorothioate modification. Cleavage activity can be retained by limiting modification to the termini and lengthening the recognition arms. Such DNA ribonucleases can be made to specifically cleave target HBV RNA and substantially inhibit intracellular viral gene expression.

Base Sequence↗
PubMed1995

Ultraviolet light-responsive element (TGACAACA)-binding proteins in cells of xeroderma pigmentosum patients.

The ultraviolet light (UV)-responsive element (URE) is an octamer sequence, TGACAACA, that shares homology with cyclic AMP-responsive element and activator protein 1 target sequences. Because URE-binding proteins have been shown to play a role in cellular response to DNA damage, we determined their expression and DNA-binding activities in repair-deficient cells. Of the complementation groups tested, only xeroderma pigmentosum (XP)-C cells induced expression of c-jun after UV irradiation; this correlated with XP-C binding to the URE and resembled the pattern observed with normal human fibroblasts. In other cases either a decrease (XP-A) or no change (XP-D) in URE-binding activities was noticed, which may be associated with decreased c-fos and poor c-jun expression after UV irradiation. That XP-C cells were the only complementation group exhibiting URE-binding activities similar to those of repair-proficient cells points to the possible correlation between proper repair of transcriptionally active genes and the expression and activities of proteins implicated in the cellular response to UV irradiation.

Base Sequence↗
PubMed1996

The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae.

CCE1 is a DNA junction-resolving enzyme involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. The CCE1 gene was cloned by PCR, and the expressed protein purified to homogeneity. CCE1 was found to bind to four-way DNA junctions, with a strong structural selectivity. The enzyme binds DNA junctions as a dimer, with slow subunit exchange occurring in free solution. While CCE1 binds equally to synthetic four-way DNA junctions of any sequence, it exhibits pronounced sequence-selectivity in cleavage. Both fixed junctions and those capable of branch migration can be cleaved, with a preference for cleavage at the sequence 5'CT/. Cleavage of junctions tethered to adopt specific stacking isomers demonstrated that the target sequences are cleaved fivefold faster when located on a continuous strand compared to an exchanging strand.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding, fungal infection and the elicitor chitosan.

The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chi1. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.

Amino Acid Sequence↗
PubMed1987

The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing.

The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis. The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich. The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA. Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.

Base Sequence↗
PubMed2000

Genomic structure and chromosomal localization of the gene encoding TRAX, a Translin-associated factor X.

The TRAX gene encodes a Translin-associated 33-kDa protein partner, TRAX. The TRAX protein has extensive amino acid homology with Translin, and contains bipartite nuclear targeting sequences, suggesting a possible role in the selective nuclear transport of Translin lacking any nuclear targeting motifs. In the present study, genomic clones of the human TRAX gene were isolated to determine the complete genomic organization. The genomic structure of the human TRAX gene was similar to that of the human Translin gene, consisting of six exons and five introns, encompassing approximately 27kb in genomic DNA. Northern blot analysis revealed a predominant transcript of approximately 2.7kb, and its distribution in various tissues was like that of Translin. Chromosomal mapping by fluorescence in situ hybridization (FISH) analysis allowed localization of the TRAX gene to human chromosome lq41.

Carrier Proteins↗
PubMed1991

Alterations in the expression of muscle-specific genes mediated by troponin C antisense oligodeoxynucleotide.

The effectiveness of an antisense oligodeoxynucleotide to troponin C (TnC) mRNA in blocking expression of TnC in differentiated chicken myotubes was examined. An 18-nucleotide-long sequence common to both fast and slow isoforms of TnC mRNAs was chosen as the target sequence. The oligomer was found to be efficiently taken up by myotubes. However, the intracellular half-life of the oligomer was found to be only 3 h. Results of studies using different concentrations of oligomer for 3 h in the culture medium showed that compared to the untreated control culture, myotubes incubated with 20 microns antisense oligomer showed a 30% reduction in the steady-state level of TnC mRNAs. An increase of incubation period to 12 h with additions of fresh culture medium containing 20 microns antisense oligomer every 3 h failed to produce any further reduction of TnC mRNA level. Concomitant to the decrease of TnC mRNAs in antisense oligomer-treated cells, the steady-state levels of alpha-actin and alpha-tropomyosin mRNAs were also reduced by approximately 20 to 40%. Analysis of the homology of the sense sequence of this oligomer with that of alpha-actin and alpha-tropomyosin mRNAs suggested that reduction in the level of alpha-actin and alpha-tropomyosin mRNAs was not due to direct hybridization of the antisense oligomer to these mRNAs. Comparison of TnC polypeptide synthesis in untreated and oligomer-treated myotubes showed approximately 70% reduction of fast TnC polypeptide synthesis in antisense oligomer-treated cells. In contrast, slow TnC polypeptide synthesis was not significantly reduced in treated cells. Similarly, alpha-actin and alpha-tropomyosin polypeptide synthesis remained close to the level of untreated cells. Furthermore, analysis of transcription of various muscle-specific mRNAs showed increased synthesis of both TnC and alpha-tropomyosin mRNAs in antisense oligomer-treated myotubes. On the other hand, synthesis of actin mRNAs was not altered by this treatment. These results showed that antisense oligomer was effective in significantly reducing TnC polypeptide synthesis in chicken myotubes. Furthermore, these results suggest that treatment of myotubes with antisense oligomer to TnC mRNA may have triggered a complex array of compensatory processes.

Actins↗
PubMed1995

Cloning a cDNA from human NK/T cells which codes for a protein with high proline content.

A cDNA clone, B4-2, was isolated from a natural killer (NK) minus T cell subtractive library. The B4-2 clone coded for an mRNA of 2061 bp in length. It encodes a deduced 327 aa protein with a calculated molecular mass of 35.2 kDa. Searching of B4-2 DNA and protein sequences against various databases revealed no high homology to other sequences. However, B4-2 has an unusually high proline content (13%), contains a putative nuclear targeting sequence, and has several SPXX motifs which are frequently found in gene regulatory proteins. One of the stretches of prolines in B4-2 closely resembles the ligand for proteins with SH3 domains. Northern hybridization data showed that B4-2 is not a lymphoid specific gene and is expressed in a hepatoma cell line and also weakly transcribed or absent in a variety of other cells. A polyclonal antiserum raised against recombinant B4-2 recognizes a 32-34 kDa protein in lymphocytes.

Amino Acid Sequence↗
PubMed2006

Rapid identification of Enterobacteriaceae by sequencing DNA gyrase subunit B encoding gene.

Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioMérieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.

Bacterial Typing Techniques↗
PubMed1998

Artificial nine zinc-finger peptide with 30 base pair binding sites.

It is well-known that DNA binding of native nine zinc-finger protein TEIIIA is dominated by interaction of select few fingers. Newly designed zinc-finger peptide Sp1ZF9 containing nine Cys2-His2 type motifs has been manipulated. The DNA-binding property of Sp1ZF9 was compared with those of native three zinc-finger Sp1(530-623) and artificial six zinc-finger Sp1ZF6 peptides. Although the equilibrium time was less than 0.5 h for Sp1(530-623)-DNA complex, Sp1ZF6 and Sp1ZF9 required approximately 48 and 72 h, respectively, for full complex formation. Evidently, the footprinting analysis demonstrated that Sp1ZF9 and Sp1ZF6 bind at least 27 and 18 contiguous base pairs of DNA sequence, respectively. Sp1ZF9 showed two step bindings to DNA, namely first the recognition of GC (5'-GGG-GCG-GGGCC-3') sequence by the N-terminal Sp1 domain and next the recognition of the corresponding target sequences by the middle and C-terminal Sp1 domains. In contrast with unimolecular binding of Sp1ZF9 and Sp1ZF6, two Sp1(530-623) molecules bind to one GCIII (5'-GGG-GCG-GGG-GGG-GCG-GGG-GGG++ +-GCG-GGGCC-3') site region. Semispecific complex formed at the beginning of Sp1ZF9-DNA interaction has also been characterized by kinetic analysis using surface plasmon resonance. Interestingly, the association rate constants for GC and GCIII complexes of Sp1ZF9 are smaller than those of the corresponding Sp1(530-623) complexes. Of special interest is the fact that new nine zinc-finger peptide Sp1ZF9 can bind to DNA sequence of approximately 30 base pairs. Such multi zinc-finger peptides may be useful as genome-specific transcriptional switches in future.

Base Pairing↗
PubMed2006

A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4.

SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an alpha1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.

Adenoma↗
PubMed1998

RP58 associates with condensed chromatin and mediates a sequence-specific transcriptional repression.

An approximately 120-amino acid domain present generally at the NH2 termini, termed the POZ domain, is highly conserved in various proteins with zinc finger DNA binding motifs. We have isolated a novel protein sharing homology with the POZ domain of a number of zinc finger proteins, including the human BCL-6 protein. By using a binding site selection technique (CAST), a high affinity binding site of the protein was determined to be (A/C)ACATCTG(G/T)(A/C), containing the E box core sequence motif. The protein was shown to repress transcription from a promoter linked to its target sequences and was hence named RP58 (Repressor Protein with a predicted molecular mass of 58 kDa). Immunogold electron microscopic study revealed that almost all RP58 is localized in condensed chromatin regions. These observations demonstrate for the first time that a protein mediating a sequence-specific transcriptional repression associates with highly condensed chromatin. We suggest that RP58 may be involved in a molecular link between sequence-specific transcriptional repression and the organization of chromosomes in the nucleus.

Amino Acid Sequence↗
PubMed1993

Murine Pax-2 protein is a sequence-specific trans-activator with expression in the genital system.

The murine paired box containing gene Pax-2 has been proposed to be involved in kidney and central nervous system (CNS) development. In this report, we show that expression cloning of Pax-2 cDNA allowed in vitro identification of specifically bound DNA sequences. When fused to the thymidine kinase (TK) promoter in front of reporter genes, these target sequences were able to mediate trans-activation by Pax-2 protein, thus demonstrating their in vivo function. Expression studies from adult mouse tissues revealed high levels of Pax-2 transcripts in male and female genital tracts, suggesting a second phase of Pax-2 activity. Sequence-specific DNA binding and subsequent modulation of promoter activities may constitute the molecular mechanism of Pax-2 action in specific adult tissues and during development.

Animals↗
PubMed1998

High-resolution analysis of cytosine methylation in the 5ĺong terminal repeat of retroviral vectors.

Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.

Animals↗
PubMed1994

High sensitivity mapping of methylated cytosines.

An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.

5-Methylcytosine↗
PubMed1995

A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151.

Binding Sites↗