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PubMed1990

Inter- and intraspecific heterochromatin variation detected by restriction endonuclease digestion in two sibling species of the Anopheles maculipennis complex.

The sibling species Anopheles atroparvus and Anopheles labranchiae are cytogenetically almost indistinguishable. The chromosome complement (2n = 6) consists of two pairs of autosomes and two heteromorphic sex chromosomes with largely homologous heterochromatic long arms. Treatment of chromosome preparations with the restriction endonucleases, Alu I, Hae III, Mbo I, Hpa II, revealed species-specific differences of the sex chromosome banding pattern. These differences involved both amount and location of digested heterochromatin. Heterochromatin heterogeneity and a high level of intraspecific polymorphism, undetected with standard banding techniques, were observed in both species. Quantitative heterochromatin differences between the sex chromosomes did not inhibit their pairing and chiasmata formation. The endonuclease Msp I, which cleaves the same target sequence as Hpa II, did not digest heterochromatic as well as euchromatic regions in both species: inhibition of cleavage by methylation of the target sequence or limited access of the enzyme to the target could be involved in this response.

Animals↗
PubMed1999

Improvement of the immune response against plasmid DNA encoding OspC of Borrelia by an ER-targeting leader sequence.

The present study outlines the characterization of a DNA-based immune response against the OspC antigen, one of the most promising candidates for a Borrelia vaccine. Balb/c mice were injected intradermally with plasmid DNA encoding the OspC gene (lacking the natural leader sequence) under transcriptional control of the cytomegalovirus (CMV) promotor. Immunization with this construct elicited only a marginal response, which was drastically improved by a fusion construct containing the human tissue plasminogen activator (hTPA) signal sequence. The results indicate that for DNA-based immunization against OspC an ER-targeting signal may be necessary for both antibody production as well as cellular immune responses.

Amino Acid Sequence↗
PubMed1987

The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases DpnII and dam from E. coli and phage T4.

The amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5'-adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC. Despite large differences on the DNA level, the four sequences show four blocks of homologies. One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA methyltransferases. It is speculated that it could be the binding site of the methyl donor, S-adenosylmethionine. On the other hand, the identification of a DNA-binding region is more tenuous. As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix-turn-helix motif have been observed.

Amino Acid Sequence↗
PubMed1997

Mft52, an acid-bristle protein in the cytosol that delivers precursor proteins to yeast mitochondria.

We have identified a novel protein, Mft52, in the cytosol of yeast cells. Mft52 has a two-domain structure that includes a receptor-like carboxyl-terminal "acid-bristle" domain, which binds basic, amphipathic mitochondrial targeting sequences. Native Mft52, purified from the cytosol of yeast cells, is found as a large particle eluting in the void volume of a Superose 6 gel filtration column. Fusion proteins, consisting of mitochondrial targeting sequences fused to nonmitochondrial passenger proteins, are targeted to mitochondria in wild-type yeast cells, but defects in the gene encoding Mft52 drastically reduce the delivery of these proteins to the mitochondria. We propose that Mft52 is a subunit of a particle that is part of a system of targeting factors and molecular chaperones mediating the earliest stages of protein targeting to the mitochondria.

Amino Acid Sequence↗
PubMed1996

Effect on DNA transcription of nucleotide sequences upstream to T7 promoter.

The T7 RNA polymerase-dependent transcription was studied as a function of nucleotide sequence structures positioned upstream of the T7 promoter. Model double-stranded DNA templates were constructed for this purpose. They contained a target sequence of 485 base pairs (cDNA fragment of Venesuelian encephalomyelitis equine virus genome), T7 promoter consensus and different extra base sequences upstream of the T7 promoter. The level of the target sequence transcription was clearly determined by the extra base sequence. The presence of one extra base pair G.C ensured the most pronounced effect, transcription was increased one order of magnitude in comparison with template which has only a canonical T7 promoter sequence at the 5'-end.

Base Sequence↗
PubMed2005

Bangladeshi medicinal plant extracts inhibiting molecular interactions between nuclear factors and target DNA sequences mimicking NF-kappaB binding sites.

Several medicinal plants can be employed to produce extracts exhibiting biological effects. The aim of this work was to verify the ability of extracts derived from different medicinal plants of Bangladesh in interfering with specific DNA-protein interactions. The rationale for this study is based on the observation that alteration of gene transcription represents a very promising approach to control the expression of selected genes and could be obtained using different molecules acting on the interactions between DNA and transcription factors (TFs). We have analysed the antiproliferative activity of extracts from the medicinal plants Hemidesmus indicus, Polyalthia longifolia, Aphanamixis polystachya, Moringa oleifera, Lagerstroemia speciosa, Paederia foetida, Cassia sophera, Hygrophila auriculata and Ocimum sanctum. Antiproliferative activity was assayed on different human cell lines, including erythroleukemia K562, B-lymphoid Raji, T-lymphoid Jurkat and erythroleukemia HEL cell lines. We employed the electrophoretic mobility shift assay (EMSA) as a suitable technique for the identification of plant extracts altering the binding between transcription factors and the specific DNA elements. We found that low concentrations of Hemidesmus indicus, Polyalthia longifolia, Moringa oleifera and Lagerstroemia speciosa, and very low concentrations of Aphanamixis polystachya extracts inhibit the interactions between nuclear factors and target DNA elements mimicking sequences recognized by the nuclear factor kappaB (NF-kappaB). On the contrary, high amount of extracts from Paederia foetida, Cassia sophera, Hygrophila auriculata or Ocimum sanctum were unable to inhibit NF-kappaB/DNA interactions. Extracts inhibiting both NF-kappaB binding activity and tumor cell growth might be a source for anti-tumor compounds, while extracts inhibiting NF-kappaB/DNA interactions with lower effects on cell growth, could be of interest in the search of compounds active in inflammatory diseases, for which inhibition of NF-kappaB binding activity without toxic effects should be obtained.

Antineoplastic Agents↗
PubMed1997

Quantitation of parvovirus B19 DNA sequences by competitive PCR: differential hybridization of the amplicons and immunoenzymatic detection on microplate.

A competitive PCR assay was developed to quantify B19 DNA sequences. Target and internal standard sequences were co-amplified by the same set of primers. The internal standard competitor was constructed by recombinant PCR and differed from the original genome sequence in a 21-bp mutagenized fragment, internal to the region amplified by the same set of primers. The internal standard competitor was cloned in a plasmid vector and the cloned fragment used in all the experiments. Target and internal standard sequences were labelled with digoxigenin during the co-amplification reaction and the different amplicons were detected in two separate hybridization reactions by biotinylated probes specific for the original 21-bp sequence or the mutagenized one. Hybridized amplicons were captured onto streptavidin-oated microtitre wells and detected by anti-digoxigenin antibodies conjugated to peroxidase. The chromogenic reaction for peroxidase was quantitatively evaluated by optical density determination. The titration curve subsequently developed showed a linear relationship over the range 10(2) to 10(5) genome copies, thus obtaining an exact quantitative evaluation over a wide range together with good sensitivity. Nine reference serum samples positive for B19 DNA and eight negative serum samples were tested by the competitive PCR assay for the quantitation of B19 DNA sequences.

Cloning, Molecular↗
PubMed1996

Interaction of a potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting determinants.

A protein of 80 kD from developing pea (Pisum sativum) cotyledons has previously been shown to exhibit characteristics of a vacuolar targeting receptor by means of its affinity for the amino-terminal vacuolar targeting sequence of proaleurain from barley (Hordeum vulgare). In this report we show that the same protein also binds to the amino-terminal targeting peptide of prosporamin from sweet potato (Ipomoea batatas) and to the carboxyl-terminal targeting determinant of pro-2S albumin from Brazil nut (Bertholletia excelsa). The receptor protein does not bind to the carboxyl-terminal propeptide (representing the targeting sequence) of barley lectin. The binding of the 80-kD protein to the sporamin determinant involves a motif (NPIR) that has been shown to be crucial for vacuolar targeting in vivo. The binding to the carboxyl-terminal targeting determinant of pro-2S albumin appears to involve the carboxyl-terminal propeptide and the adjacent five amino acids of the mature protein. The 80-kD protein does not bind to peptide sequences that have been shown to be incompetent in directing vacuolar targeting.

Amino Acid Sequence↗
PubMed2005

High-throughput double quantitative competitive polymerase chain reaction for determination of genetically modified organisms.

Quantitative competitive polymerase chain reaction (PCR), especially the double competitive PCR methods (DC-PCR), have evolved as reliable approaches to quantification of genetically modified organisms (GMO) in food. However, DC-PCR is a low-throughput method because it requires titration of each sample with various amounts of a competitive internal standard, a protocol that involves several PCRs per sample followed by electrophoresis and densitometry. To address this drawback, we have developed a new method for GMO quantification, namely, a high-throughput double quantitative competitive PCR (HT-DCPCR). In HT-DCPCR, electrophoresis and densitometry are replaced by a rapid, microtiter well-based bioluminometric hybridization assay and there is no need for titration of each sample. The determination of GM soya was chosen as a model. We have constructed internal standards (DNA competitors) both for the 35S promoter sequence and for a plant-specific reference gene (lectin). The competitors have identical size and share the same primer binding sites with the target sequences but differ in a 24-bp internal segment. Each target sequence (35S and lectin) is coamplified with a constant amount (1000 copies) of the respective competitor. The four amplified fragments are hybridized with specific probes and captured on a universal solid phase to achieve simplicity and high throughput. The hybrids are determined by using streptavidin conjugated to the photoprotein aequorin. The ratio of the luminescence values obtained for the target and the competitor is linearly related to the starting amount of target DNA. The limit of quantification for the 35S promoter is 24 copies. The proposed method was evaluated by determining the GMO content of soybean powder certified reference materials. Also HT-DCPCR was compared to real-time PCR in a variety of real samples.

Calibration↗
PubMed1988

Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation.

A photocrosslinking reagent (p-azidophenacyl) was covalently linked to an octathymidylate synthesized with either the natural (beta) anomer of thymidine or the synthetic (alpha) anomer. The oligothymidylate was further substituted by an acridine derivative to stabilize the hybrid formed with a complementary octadeoxyadenylate sequence via intercalation. A single-stranded 27-mer containing a (dA)8 sequence and a 27-mer duplex containing a (dA.dT)8 sequence were used as targets. Upon UV irradiation, photocrosslinking of the octathymidylate to its target sequence was observed, generating bands that migrated more slowly in denaturing gels. In the 27-mer duplex, both strands were photocrosslinked to the octathymidylate. Upon alkaline treatment of the irradiated samples, cleavage of the 27-mers was observed at specific sites. These reactions were analyzed at different salt concentrations. The location of the cleavage sites allowed us to demonstrate the following. (i) Both alpha and beta oligothymidylates can recognize a DNA double helix containing an oligo(dA).oligo(dT) sequence; the oligothymidylate binds to the major groove of DNA in a parallel orientation with respect to the adenine-containing strand of the DNA double helix. (ii) alpha oligothymidylates form helices with a complementary single-stranded oligodeoxyadenylate; the two strands have a parallel orientation independently of whether or not an intercalating agent is attached to the oligothymidylate. (iii) At low salt concentration, beta oligothymidylates form a double helix with an oligodeoxyadenylate in which, as expected, the two strands are antiparallel; at high salt concentration, a triple helix is formed in which the second oligothymidylate is oriented parallel to the adenine-containing strand. These results show that it is possible to recognize an oligopurine.oligopyrimidine sequence in a DNA double helix via local triple-helix formation and to target photochemical reactions to specific sequences in both double-stranded and single-stranded nucleic acids.

Cross-Linking Reagents↗
PubMed2006

Small interfering RNAs targeting mutant K-ras inhibit human pancreatic carcinoma cells growth in vitro and in vivo.

AIM: To investigate the effect of small interfering RNAs targeting mutant K-ras on the growth of pancreatic carcinoma cell lines in vitro and in vivo. MATERIALS AND METHODS: We cloned targeting sequence spanning codon 12 of mutant K-ras into the pSilencer-hygro plasmid, yielding two recombinant vectors with one base different. Both human pancreatic carcinoma cell lines were transfected by these two recombinant vectors. The transfected PC-7 cells were injected subcutaneously into nude mice to observe its tumorigenicity. RT-PCR and Western blot analysis were carried out to test the expression of K-ras in all of the transfected cell lines. Growth curves assay were performed to test the abilities of cells proliferation. Anti-K-ras therapy of PC-7 and Panc-1 in subcutaneous mice models were performed by intratumor injection of polyethylenimine/siRNAs complex. RESULTS: The expressions of K-ras in PC-7 cells and Panc-1 cells were significantly inhibited by corresponding small interfering RNAs. The expression of K-ras was particularly inactivated by siRNA without any base mismatch to its homologous mRNA, while this oncogene with central base mismatch could not be inhibited as effectively as that of the former. The growth of PC-7 cells and Panc-1 cells transfected by corresponding mutant K-ras targeted siRNAs were significantly suppressed when compared with controls (p<0.05). The transfected PC-7 cells lost tumorigenic ability. Four weeks treatment of Xenograft of pancreatic carcinoma (PC-7 and Panc-1) in nude mice with Polyethylenimine-encapsulated mutant K-ras targeted siRNAs (20 microg/mouse twice weekly) were effective in reducing tumor growth, when compared with controls (p<0.05). CONCLUSION: The central base may play a key role in the process of RNA interference. The mutant point and its vicinity of 19 nucleotides in K-ras may be the effective targeting sequence for RNA interference. Targeting mutant-k-ras therapy of pancreatic carcinoma may be a clinically applicable therapeutic modality.

Cell Division↗
PubMed1995

Distinct CG and CNG DNA methyltransferases in Pisum sativum.

DNA methyltransferase activity, present in low salt extracts of nuclei from young pea shoot apices, has been fractionated into two different species by assaying with model substrates. The CG methyltransferase (an unstable enzyme believed to be of 140 kDa) methylates cytosine only in oligonucleotides with CG and Cl dinucleotide targets while an enzyme of 110 kDa (the CNG methyltransferase) methylates the cytosines in 5'-CAG-3' and 5'-CTG-3' target sequences, especially when hemimethylated, but not in 5'-CCG-3' nor in 5'-CGG-3' target sequences present in oligonucleotides.

Base Sequence↗
PubMed2006

A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells.

Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siRNA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.

Animals↗
PubMed2003

Sequence-specific targeting of Drosophila roX genes by the MSL dosage compensation complex.

MSL complexes bind the single male X chromosome in Drosophila to increase transcription approximately 2-fold. Complexes contain at least five proteins and two noncoding RNAs, roX1 and roX2. The mechanism of X chromosome binding is not known. Here, we identify a 110 bp sequence in roX2 characterized by high-affinity MSL binding, male-specific DNase I hypersensitivity, a shared consensus with the otherwise dissimilar roX1 gene, and conservation across species. Mutagenesis of evolutionarily conserved sequences diminishes MSL binding in vivo. MSL binding to these sites is roX RNA dependent, suggesting that complexes become competent for binding only after incorporation of roX RNAs. However, the roX RNA segments homologous to the DNA binding sites are not required, ruling out simple RNA-DNA complementarity as the primary targeting mechanism. Our results are consistent with a model in which nascent roX RNA assembly with MSL proteins is an early step in the initiation of dosage compensation.

Animals↗
PubMed1998

Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR.

UNLABELLED: Although many genetic diseases are caused by the presence of point mutations in respective genes, an increasing number of diseases are known to be caused by gene copy number changes. We report the development of a rapid and reliable PCR-based method for quantitation of gene copy number with sufficient sensitivity to detect single copy changes without the use of radioactive or fluorescent labeling. The sensitivity of this technique has been demonstrated by the detection of the DNA duplication or deletion occurring in two inherited peripheral neuropathies, Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), that are caused by a reciprocal duplication or deletion event on chromosome 17p11.2-12. This method relies on the comparison of the amount of PCR product generated from a potentially duplicated or deleted target sequence with the amount of product generated from a disomic reference gene. The value of this ratio (target PCR product:reference PCR product) indicates whether the target sequence is duplicated, deleted, or unchanged. Using primers from within a duplicated or deleted region (PMP22 gene and EW401) and from within a reference region (NF1 gene), we tested 50 CMT1A, 30 HNPP, and 50 unaffected individuals for the presence of a DNA duplication or deletion. TARGET: reference ratios of 1.58, 1.02, and 0.56 were detected for the CMT1A, unaffected, and HNPP groups, respectively. Thus, differentiation of the three groups of individuals was on the basis of gene copy number. This technique was successfully used to detect the difference in the X chromosome copy number between males and females (target:reference ratios of 1.1 and 2.3, respectively). This approach to the detection of DNA duplications and deletions is sensitive, accurate, and has potential applications in the quantitation of changes in gene copy number associated with diseases characterized by such chromosomal alterations.

Charcot-Marie-Tooth Disease↗
PubMed2003

Sequence-specific targeting of two bases on both DNA strands by the somatic hypermutation mechanism.

Somatic mutations within antibody genes alter the affinity and selectivity of antibody molecules and largely define the quality of the memory B cell repertoire in many vertebrate species. While some evidence supports the idea that there is a strand bias to the hypermutation mechanism, conflicting data suggest that somatic mutations are initially acquired on both strands of DNA. In this study, we utilized a previously defined trinucleotide target bias of hypermutation to address the question of target strand symmetry during mutation. Mutabilities of specific base positions within all triplets were compared between the two strands of DNA in three divergent databases of hypermutated sequences. Unexpectedly, we consistently observed strong correlations between mutabilities of triplet positions on the two DNA strands only for G and T in the first position of a triplet or for C and A in the last position. The most straightforward interpretation of this result is that the mutation mechanism targets either G and T or C and A on both strands of DNA with a frequency that depends upon the adjacent dinucleotide sequence. In view of published evidence that C is targeted by the hypermutation mechanism, we can extrapolate that C and A are specifically targeted at a frequency that depends upon the preceding 5' dinucleotide.

Animals↗
PubMed2004

Reassessment of sequence-based targets for identification of bacillus species.

The Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevant B. cereus and B. anthracis as well as nonpathogenic B. psychrotolerans and B. psychrodurans. Consequently, 27 type and clinical strains from the B. cereus group were used to test alternate targets (rpoB, vrrA, and the 16S-23S spacer region) for identification. The rpoB gene proved the best alternate target, with a conserved 4-nucleotide difference between B. cereus and B. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database at http://www.ridom_rdna.de/.

Bacillus↗
PubMed1994

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes.

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320-322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47-dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.

Amino Acid Sequence↗