Specific Oligonucleotide Primers Research Articles

Protection against Anoikis and Down-regulation of Cadherin Expression by a Regulatable β-Catenin Protein

beta-Catenin signaling plays a key role in a variety of cellular contexts during embryonic development and tissue differentiation. Aberrant beta-catenin signaling has also been implicated in promoting human colorectal carcinomas as well as a variety of other cancers. To study the molecular and cellular biological functions of beta-catenin in a controlled fashion, we created a regulatable form of activated beta-catenin by fusion to a modified estrogen receptor (ER) ligand binding domain (G525R). Transfection of tissue culture cells with expression vectors encoding this hybrid protein allows the signal transduction function of beta-catenin to be induced by the synthetic estrogen, 4-hydroxytamoxifen, leading to regulated activation of a beta-catenin-lymphocyte enhancer-binding factor-dependent reporter gene as well as induction of endogenous cyclin D1 expression. The activation of ER-beta-catenin signaling rescues RK3E cells from anoikis and correlates with an increased phosphorylation of mitogen-activated protein kinase. The inhibition of anoikis by ER-beta-catenin can be abolished by a mitogen-activated protein kinase pathway inhibitor, PD98059. Evidence is also provided to show that ER-beta-catenin down-regulates cadherin protein levels. These findings support a key role for activated beta-catenin signaling in processes that contribute to tumor formation and progression.

Microsatellite markers and clonal genetic structure of the fungal pathogen Phialophora gregata

A microsatellite-enriched library was constructed from genomic DNA of Phialophora gregata f. sp. sojae , the causal agent of soybean brown stem rot. Specific oligonucleotide primer pairs were designed to flank individual microsatellite loci, and used in PCR to detect polymorphisms among 118 contemporary and archival isolates of P. gregata from several countries, which represent two distinct, but sympatric populations delineated by rDNA genotypes. In general, low levels of genetic variation were observed and manifested in two ways. First, the number of polymorphic loci was low. Among 24 microsatellite loci screened, four polymorphic loci were detected, and two of them were located on the same clone. Second, the number of alleles in the polymorphic loci was low. Only two alleles were observed in each polymorphic locus. Alleles of the polymorphic microsatellite loci were invariant within, but different between, the two populations of P. gregata. From all evidence available, P. gregata f. sp. sojae exhibits several genetic features characteristic of clonal genetic structure, namely widespread distribution and stability of identical genotypes, absence of recombinant genotypes, and correlation of independent genetic traits including three sets of molecular markers, defoliating and nondefoliating pathotypes, and differential pathogenic fitness.

Identification of six mRNA sequences of genes related to multixenobiotic resistance (MXR) and biotransformation in Mytilus edulis

Tissue-specific gene expression of the MXR (multixenobiotic resistance) and biotrans- formation-related gene homologs for the multidrug resistance protein 1 (Mdr 1), the multidrug resis- tance protein (Mrp), the major vault protein (Mvp, i.e. the lung resistance protein, Lrp), glutathione S-transferase (Gst pi), heat-shock protein 70 gene (Hsp70), and cytochrome p450 (Cyp4A) was analysed in the blue mussel Mytilus edulis. Additionally, Topoisomerase II (TopoII) and actin were investigated as indicators for mitotic activity and as internal standards. We identified highly con- served regions in the corresponding genes of several other species, and synthesised degenerate olignucleotide primers designed to amplify these regions from M. edulis mRNA. Resulting RT-PCR products were cloned into a T-tailed vector and DNA-sequenced. The results were evaluated by com- puter-based sequence alignment with the known gene sequences of blueprint species. PCR amplified fragments were used as probes for Northern blot hybridisation to identify transcript sizes. Specific oligonucleotide primers were designed from the M. edulis sequences for each gene and used for semi-quantitative multiplex RT-PCR to investigate tissue-specific gene expression. We have cloned and DNA-sequenced segments of the M. edulis P-gp, Mvp(Lrp), Gst pi, Hsp70, TopoII and actin genes. Semi-quantitative multiplex RT-PCR rapidly identified characteristic differential gene- expression patterns in a range of mussel tissues, and is therefore a promising tool for comprehensive studies of gene regulation in marine invertebrates in adaptation to various physiological conditions, including responses to natural and anthropogenic stressors.

Megaprimer method for polymerase chain reaction-mediated generation of specific mutations in DNA.

During the past decade, a number of methods using polymerase chain reaction (PCR) for the generation of specific mutations in any nucleotide sequence have been described, these methods include such drawbacks as the possibility of generating undesired secondary mutations, because of the low fidelity of some of the thermostable DNA polymerases, the need for four or more specific oligonucleotide primers, and the use of sophisticated, individually optimized protocols. Finally, the selection of correctly mutated clones may also prove to be laborious (, , ). In contrast, the megaprimer principle described here is costefficient, fast, reliable, and convenient.

Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.

Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.

Role of the Deafness Dystonia Peptide 1 (DDP1) in Import of Human Tim23 into the Inner Membrane of Mitochondria

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.

Identification of Sporothrix schenckii based on sequences of the chitin synthase 1 gene

Sporothrix schenckii is pathogenic to human and animals. To detect S. schenckii in the tissue, we designed specific oligonucleotide primers based on the chitin synthase gene. Amplification products were selectively obtained from S. schenckii DNA. Polymerase chain reaction analysis with the primer pair S2-R2 was able to detect 10 pg genomic DNA of S. schenckii with ethidium bromide staining. This detection system will be useful as a microbiological tool for the diagnosis of human and animal sporotrichosis.

Isolation of an insertion sequence from Ralstonia solanacearum race 1 and its potential use for strain characterization and detection.

A new insertion sequence (IS), IS1405, was isolated and characterized from a Ralstonia solanacearum race 1 strain by the method of insertional inactivation of the sacB gene. Sequence analysis indicated that the IS is closely related to the members of IS5 family, but the extent of nucleotide sequence identity in 5' and 3' noncoding regions between IS1405 and other members of IS5 family is only 23 to 31%. Nucleotide sequences of these regions were used to design specific oligonucleotide primers for detection of race 1 strains by PCR. The PCR amplified a specific DNA fragment for all R. solanacearum race 1 strains tested, and no amplification was observed with some other plant-pathogenic bacteria. Analysis of nucleotide sequences flanking IS1405 and additional five endogenous IS1405s that reside in the chromosome of R. solanacearum race 1 strains indicated that IS1405 prefers a target site of CTAR and has two different insertional orientations with respect to this target site. Restriction fragment length polymorphism (RFLP) pattern analysis using IS1405 as a probe revealed extensive genetic variation among strains of R. solanacearum race 1 isolated from eight different host plants in Taiwan. The RFLP patterns were then used to subdivide the race 1 strains into two groups and several subgroups, which allowed for tracking different subgroup strains of R. solanacearum through a host plant community. Furthermore, specific insertion sites of IS1405 in certain subgroups were used as a genetic marker to develop subgroup-specific primers for detection of R. solanacearum, and thus, the subgroup strains can be easily identified through a rapid PCR assay rather than RFLP analysis.

Stage-dependent Localization of a Novel Gene Product of the Malaria Parasite, Plasmodium falciparum

A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.

TGF-beta isoform and receptor expression in giant cell tumor and giant cell lesions of bone.

The authors examined the distribution of tumor growth factor-beta (TGF-beta) isoforms and receptors in 35 giant cell tumor (GCT) of bone in comparison with a group of benign giant cell-containing lesions of bone, including 5 aneurysmal bone cysts, 2 cases of brown tumor of hyperparathyroidism, 3 nonossifying fibromas, and 7 cases of giant cell reparative granuloma. The results of immunohistochemical analysis of GCT showed a complete absence of TGF-beta1 expression in both mononuclear tumor cells and giant cells. Only reactive bone present within the tumor showed an intense immunoreactivity. Transforming growth factor-beta2 and TGF-beta3 were detected in the majority of cases (97.1% and 82.8%, respectively), whereas TGF-beta receptor type I (TGF-beta RI) and type II (TGF-beta RII) were diffusely expressed in all cases. Reverse transcription-polymerase chain reaction (RT-PCR) analysis performed on 10 GCTs with specific oligonucleotide primers demonstrated the presence of mRNA transcripts for TGF-beta1, 2, 3, and for TGF-beta RI and RII. Quantitative measurements of TGF-beta1 in conditioned media from primary cultures of GCT showed undetectable or very low amounts of the cytokine (0-23 pg/mL). The results of immunohistochemical analysis showed that all giant cell-containing lesions of bone were at least focally positive for the 3 isoform of TGF-beta, with positivity present both in osteoclast-like giant cells and mononuclear cells, and diffusely positive for TGF-beta RI and RII. Reverse transcription-polymerase chain reaction analysis conducted on samples from 3 nonossifying fibromas and 1 giant cell reparative granuloma confirmed the expression of the corresponding mRNA. In conclusion, according to the current data, GCT of bone can be distinguished from other giant cell-containing lesions of bone on the basis of the absence of TGF-beta1 expression at the protein level, which appears to be the result of posttranslational regulation processes.

Aromatase messenger RNA is derived from the proximal promoter of the aromatase gene in Leydig, Sertoli, and germ cells of the rat testis.

It has long been recognized that individual cell types within the testes possess the capacity to synthesize estrogen. A number of studies on different species have demonstrated that the levels of aromatase expression and the patterns of regulation are distinct between the different cell types of the testes. Whereas a variety of promoters have been shown to contribute to the patterns of aromatase expression in different cell lineages, studies using ovarian RNA, testis RNA, and Leydig cell tumor lines have demonstrated that the same promoter (promoter II) was used in each. Recent experiments using potent aromatase inhibitors or analysis of animals in which the genes encoding the estrogen receptor-alpha (ER-alpha) or the aromatase, P450, are defective, have confirmed the importance of local estrogen formation in normal testicular function. In order to permit experiments to identify the elements controlling aromatase expression in the individual cell compartments of the testes, we prepared RNA from purified preparations of Leydig, Sertoli, and germ cells. Using specific oligonucleotide primers, the sites of initiation of the aromatase mRNA were determined using rapid amplification of cDNA ends (RACE) and nucleotide sequence analysis of the resulting cDNA fragments. Our results indicate that aromatase mRNA is derived from the proximal promoter (PII) of the aromatase gene in each of the major cell types of the rat testes.

First evidence of a retrotransposon-like element in olive (Olea europaea): implications in plant variety identification by SCAR-marker development

When developing SCARs by sequencing RAPD markers useful for olive variety identification, one RAPD sequence of 407 bp has been identified that shows significant DNA homology with more than 160 retrotransposon-like sequences. A generally coherent phylogenetic tree has been constructed based on the homologous retrotransposon-like sequences, reflecting genetic distances in 35 species belonging to eight kingdoms. The implications of this finding in the development of SCAR markers are discussed. In addition, specific oligonucleotide primers have been developed to amplify the DNA region and have a practical application as an internal amplification control in SCAR-based multiplexed PCRs. To our knowledge, this is the first report of a retrotransposon-like element in olive.

Detection and Partial Characterization of a Second Closterovirus Associated with Little Cherry Disease, Little cherry virus-2

ABSTRACT Little cherry disease (LChD) is a complex and serious viral disease of cherry. Although originally described almost 70 years ago, there has been little progress in identifying the causal agent of the disease due to the difficulty in obtaining purified virus from infected trees. This problem was partially overcome in 1997 when the compete sequence of a closterovirus associated with LChD, Little cherry virus (LChV), was published. This virus could be associated with some, but not all, trees with LChD, indicating that another virus was also involved. We report here the partial characterization of a second closterovirus associated with LChD, Little cherry virus-2 (LChV-2), and in order to differentiate the two LChD-associated viruses, we refer to LChV as Little cherry virus-1 (LChV-1). LChV-2 is a new closterovirus with molecular similarities to Grapevine leafroll-associated virus-1 (GLRaV-1) and GLRaV-3 but only distantly related to LChV-1. Based on limited sequence comparisons, LChV-2 is the same virus previously identified in association with LChD in Canada. In reverse transcription-polymerase chain reaction detection assays using specific oligonucleotide primers to either LChV-1 or LChV-2, 27 of 28 isolates of LChD tested positive to one or both of these viruses originating from Europe and North America. These results would further confirm the association of LChV-2 with LChD. One isolate, however, tested negative to both LChV-1 and LChV-2, indicating that while this report brings us a step closer to understanding LChD, further work is required to confirm the causal agents of LChD.

Molecular characterization of the VP7 gene of Rotavirus isolated from a clinical sample of Calcutta, India

The gene coding for outer capsid protein, VP7 of rotaviruses detected among the children suffering from watery diarrhea in the eastern part of India was studied. The full length gene, coding for VP7 was synthesized from a local strain, WD33, by combined reverse transcriptase–polymerase chain reaction. A total of 42 fecal RNA out of 93 rotavirus-positive samples hybridized with this DNA, indicating the prevalence of this strain in the community. Comparison of the nucleotide and deduced amino acid sequence with the VP7 gene of other serotypes revealed that the local strain,WD33, resembled closely with G1 serotype, the homology being 94 and 97%, respectively. Multiplex PCR using the specific oligonucleotide primers also amplified the G1 specific DNA fragment. The overall change of 16 amino acids was noticed when compared with G1 specific prototype Wa strain of which nine amino acids are within the neutralization domain. However, the phylogenetic tree constructed by comparing the G1 strains from different countries demonstrated that this strain WD33 clustered as a distinct sub-lineage of lineage IV and not with Wa strain, that clustered with lineage III.

Emergence of Rotavirus G4P8 Strain among Children Suffering from Watery Diarrhea in Calcutta, India

Rotavirus RNAs from the fecal samples were hybridized with cDNAs specific for G1 and G2 genotypes. 59 out of 138 samples (42.7%) did not hybridize with either probe. The cDNAs coding for VP7 and VP4 from one such sample, SC134, were synthesized by combined reverse transcriptase/polymerase chain reaction (PCR) using specific oligonucleotide primers and were used as probes to screen those local isolates which did not hybridize with either G1 or G2. 26/59 (44%) of fecal RNA hybridized with these cDNAs indicating a possible emergence of this strain in this part of India. The VP7 and VP4 genotype specificity of SC134 was found to be G4P8 by multiplex PCR. The VP7 gene of SC134 was cloned and characterized in detail by restriction enzyme digestion and DNA sequence analysis. Comparison of nucleotide and predicted amino acid sequence of the VP7 gene of SC134 with other serotypes revealed that the VP7 gene of SC134 was closely related to G4. However, amio acids within the VP7 sequence differed in seven positions with that of both the subtype ‘A’ and ‘B’ of the G4 serotype. To establish the relation of this emerging strain with the other reported G4 strains, a phylogenetic tree was constructed and SC134 was found to be more closely related to the ST3 strain isolated in England and included in the tetravalent vaccine formulation along with two Japanese strains, although all four were distinct and did not form any cluster as was evident by the horizontal distance separating them. The VP7 gene sequence of SC134 was submitted to EMBL and was assigned the accession number AJ278217.

Identification of Candida dubliniensis Based on the Specific Amplification of Mitochondrial Cytochrome b Gene.

Candida dubliniensis, a recently described Candida species, is frequently isolated from oral candidiasis in human immunodeficiency virus infected individuals. To detect the organism rapidly, we have developed specific oligonucleotide primers based on the sequences of mitochondrial cytochrome b gene. These primers selectively amplified DNA only from C. dubliniensis; the DNAs of all pathogenic Candida species tested, as well as those of medically relevant yeasts such as Cryptococcus neoformans, and Trichosporon cutaneum, were not amplified. This is the first report describing the effectiveness of cytochrome b gene in PCR based detection of an organism, and we hope the system will be useful as a microbiological tool for rapid detection of C. dubliniensis.

Identification of periodontal pathogens in atheromatous plaques.

Recent studies suggest that chronic infections including those associated with periodontitis increase the risk for coronary vascular disease (CVD) and stroke. We hypothesize that oral microorganisms including periodontal bacterial pathogens enter the blood stream during transient bacteremias where they may play a role in the development and progression of atherosclerosis leading to CVD. To test this hypothesis, 50 human specimens obtained during carotid endarterectomy were examined for the presence of Chlamydia pneumoniae, human cytomegalovirus, and bacterial 16S ribosomal RNA using specific oligonucleotide primers in polymerase chain reaction (PCR) assays. Approximately 100 ng of chromosomal DNA was extracted from each specimen and then amplified using standard conditions (30 cycles of 30 seconds at 95 degrees C, 30 seconds at 55 degrees C, and 30 seconds at 72 degrees C). Bacterial 16S rDNA was amplified using 2 synthetic oligonucleotide primers specific for eubacteria. The PCR product generated with the eubacterial primers was transferred to a charged nylon membrane and probed with digoxigenin-labeled synthetic oligonucleotides specific for Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, and Prevotella intermedia. Eighty percent of the 50 endarterectomy specimens were positive in 1 or more of the PCR assays. Thirty-eight percent were positive for HCMV and 18% percent were positive for C. pneumoniae. PCR assays for bacterial 16S rDNA also indicated the presence of bacteria in 72% of the surgical specimens. Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 44% of the 50 atheromas were positive for at least one of the target periodontal pathogens. Thirty percent of the surgical specimens were positive for B. forsythus, 26% were positive for P. gingivalis, 18% were positive for A. actinomycetemcomitans, and 14% were positive for P. intermedia. In the surgical specimens positive for periodontal pathogens, more than 1 species was most often detected. Thirteen (59%) of the 22 periodontal pathogen-positive surgical specimens were positive for 2 or more of the target species. Periodontal pathogens are present in atherosclerotic plaques where, like other infectious microorganisms such as C. pneumoniae, they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.

Heightened Vascular Reactivity Following Sympathetic Denervation Correlates with Increased Expression of C-Kit and Connexin43

P71 Short-term denervation produces supersensitivity to α-adrenergic agonists that resembles the heightened vascular reactivity observed in hypertensive subjects. Gap junctions, transmembrane channels responsible for cell-to-cell communication, may play a role in maintaining increased vascular reactivity. We examined the effects of denervation with intra-peritoneal reserpine 3 mg/kg/day (N=5) or topical 5% phenol-glycerol (N=7) on agonist-induced oscillatory activity. Wistar-Kyoto (WKY) rat tail arteries were exposed to step-wise addition of norepinephrine (NE) 10 -9 M-10 -5 M. The concentration for half-maximal response for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (log EC 50 [mol]/L control -6.89 vs reserpine -7.16, p<0.05); control -6.86 vs phenol -7.32, p<0.0001). Incubation in K + -free solution produced an 8-fold increase in isometric tension in control vs denervated vessels (138.4±23.2 vs 17.2±4.5 % of contraction to phenylephrine 10 -6 M, p<0.001). These findings are consistent with adrenergic denervation. Agonist-induced oscillatory activity was observed in 5/5 reserpine- and 4/7 phenol-treated vessels vs 0/12 controls (Fischer’s exact test p<0.05). The highly selective gap junction inhibitor, Gap27 (10 mM), nearly abolished NE-induced vascular smooth muscle oscillations. Reverse transcriptase polymerase chain reaction was carried out for connexin43 (Cx43) and c-Kit, a marker for non-neural interstitial cells that serve as pacemaker cells, using specific oligonucleotide primers. Following denervation, mRNA levels of Cx43 remained unchanged while levels of c-Kit increased markedly (0.16±0.03 vs 0.62±0.13 a.u., p<0.05). Western blot analysis revealed near doubling of Cx43 protein after denervation (80.0±13.7 vs 41.4±5.7 a.u., p<0.05). In conclusion, sympathetic denervation produced increased agonist-induced oscillatory contractile activity in vascular smooth muscle. The heightened vascular reactivity may result from upregulation of pacemaker cells and increased Cx43 expression.

Excision from tRNA genes of a large chromosomal region, carrying avrPphB, associated with race change in the bean pathogen, Pseudomonas syringae pv. phaseolicola.

Pseudomonas syringae pv. phaseolicola (Pph) race 4 strain 1302A carries avirulence gene avrPphB. Strain RJ3, a sectoral variant from a 1302A culture, exhibited an extended host range in cultivars of bean and soybean resulting from the absence of avrPphB from the RJ3 chromosome. Complementation of RJ3 with avrPphB restored the race 4 phenotype. Both strains showed similar in planta growth in susceptible bean cultivars. Analysis of RJ3 indicated loss of > 40 kb of DNA surrounding avrPphB. Collinearity of the two genomes was determined for the left and right junctions of the deleted avrPphB region; the left junction is approximately 19 kb and the right junction > 20 kb from avrPphB in 1302A. Sequencing revealed that the region containing avrPphB was inserted into a tRNALYS gene, which was re-formed at the right junction in strain 1302A. A putative lysine tRNA pseudogene (PsitRNALYS) was found at the left junction of the insertion. All tRNA genes were in identical orientation in the chromosome. Genes near the left junction exhibited predicted protein homologies with gene products associated with a virulence locus of the periodontal pathogen Actinobacillus actinomycetemcomitans. Specific oligonucleotide primers that differentiate 1302A from RJ3 were designed and used to demonstrate that avrPphB was located in different regions of the chromosome in other strains of Pph. Deletion of a large region of the chromosome containing an avirulence gene represents a new route to race change in Pph.

Matrix Metalloproteinases Cleave Tissue Factor Pathway Inhibitor: EFFECTS ON COAGULATION

Matrix Metalloproteinases Cleave Tissue Factor Pathway Inhibitor: EFFECTS ON COAGULATION