PCR-amplified DNA Fragments Research Articles

Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.

A novel deletion in TNNI2 causes distal arthrogryposis in a large Chinese family with marked variability of expression

Distal arthrogryposis (DA) is composed of a group of clinically and genetically heterogeneous disorders, characterized by multiple congenital contractures of the limbs. Point mutations in three genes encoding contractile fast-twitch myofibers, TPM2, TNNI2 and TNNT3, were recently identified in DA type 1 (DA1; MIM 108120) and DA type 2B (DA2B; MIM 601680). We have described a large Chinese DA family in which different individuals had phenotypes similar to DA1 or DA2B. To map the disease locus in this family, two-point linkage analysis was first performed using microsatellite markers selected from the genomic regions close to the TPM2, TNNI2/TNNT3 and TNNC2 genes. A positive LOD score of 3.61 at theta = 0 was obtained with the marker close to the TNNI2/TNNT3 genes, corresponding to the genetic mapping site of DA2B. Direct sequencing of the PCR-amplified DNA fragment spanning exon 8 of the TNNI2 gene showed a heterozygous deletion, c.523_525delAAG (p.K175del), in the proband. This novel mutation was confirmed to cosegregate with the DA phenotype in affected individuals but not detected in all unaffected individuals of the family and not in 50 healthy controls. In summary, we have found a novel TNNI2 mutation in a Chinese family with DA2B. Our work represents the first report on the link between TNNI2 and the DA phenotype in Chinese.

Correlation between thymidylate synthase (TS) genotype, clinical outcome, and TS mRNA expression in subjects treated with oxaliplatin (OX) and capecitabine (Cape)

13073 Background: TS mRNA has a unique tandemly repeated 28-base pair sequence in the 5’ UTR & is polymorphic in the number of this repeat (2 or 3). Homozygosity for the 3-repeat sequence has been associated with increased TS protein content in tumor samples, & decreased likelihood of clinical benefit with TS-directed therapy. This study assessed the possible association of TS genotype with clinical outcome in subjects receiving OX &Cape in an IRB-approved Phase I clinical trial. Methods: Pre-therapy genomic DNA was isolated from peripheral blood mononuclear cells in 88 subjects treated with OX 130 mg/m2 day 1 & Cape 600–1500 mg/m2 twice daily days 1–5 & 8–12 every 3-weeks. The number of tandem repeats was determined by the size of the PCR-amplified DNA fragments by staining after 3% agarose gel electrophoresis. The genotype was also compared to TS mRNA expression that was previously determined by quantiitative real-time PCR in microdissected specimens of metastatic tumor tissue. Results: The majority of subjects participating in this trial had colorectal cancer, & most had prior 5-FU & irinotecan (median of 2 prior chemotherapies). The distribution of TS genotypes among 84 informative samples was 2/2, 12%; 2/3, 70%; 3/3, 18%. The objective response rate (CR + PR) according to genotype was 22% (2/2), 21% (2/3) & 13% (3/3). No appreciable difference was seen in the median time to treatment failure (150 vs 156 vs 156 days) or median survival (379 vs 323 vs 332 days) among the 2/2, 2/3 or 3/3 genotype cohorts. There was no significant difference in the distribution of TS intratumoral gene expression according to TS genotype: median (25th & 75th percentiles): 2/2, 3.96 (2.93, 5.3); 2/3, 4.11 (2.52, 6.62); & 3/3, 2.66 (1.73, 5.23). Conclusions: In this study involving mostly pre-treated subjects with colorectal cancer receiving OX/Cape, those homozygous for 3-tandem repeats in the 5’ UTR of TS did not experience a significantly worse clinical outcome. There was no relation between TS genotype in genomic DNA & TS mRNA expression in tumor tissue; this observation is consistent with information suggesting that the 3-repeat sequence of TS mRNA increases translational efficiency. [Table: see text]

Involvement of Topoisomerase III in Telomere-Telomere Recombination

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative (ALT) recombination mechanism. In yeast, Sgs1p and its associated type IA topoisomerase, Top3p, may work coordinately in removing Holliday junction intermediates from a crossover-producing recombination pathway. Previous studies have also indicated that Sgs1 helicase acts in a telomere recombination pathway. Here we show that topoisomerase III is involved in telomere-telomere recombination. The recovery of telomere recombination-dependent survivors in a telomerase-minus yeast strain was dependent on Top3p catalytic activity. Moreover, the RIF1 and RIF2 genes are required for the establishment of TOP3/SGS1-dependent telomere-telomere recombination. In human Saos-2 ALT cells, human topoisomerase IIIalpha (hTOP3alpha) also contributes to telomere recombination. Strikingly, the telomerase activity is clearly enhanced in surviving si-hTOP3alpha Saos-2 ALT cells. Altogether, the present results suggest a potential role for hTOP3alpha in dissociating telomeric structures in telomerase-deficient cells, providing therapeutic implications in human tumors.

Sensitive detection of a phytoplasma in Catharanthus roseus L. (G.) Don

Catharanthus roseus L. (G.). Don., a high-valued medicinal plant, is severely affected by phytoplasmas present in the sieve tubes of the phloem. The infected plant shows malformation of leaves and flowers as well as virescence of flowers. As a first step to regenerate phytoplasma-free C. roseus plants through in vitro culture, a nested-PCR based sensitive system was standardized to detect phytoplasma from various parts of naturally as well as in vitro propagated phytoplasma-infected plants. Universal primers, from the 16S rDNA and the 16S—23S rDNA spacer region sequences of phytoplasma, were applied in direct/nested-PCR. Total DNA extracts from different parts of naturally infected as well as in vitro propagated phytoplasma-infected plants were subjected to direct PCR. The direct PCR products were subsequently used as templates in nested PCR. It yielded a DNA fragment of 1.2 kb representing the 16S rDNA of phytoplasma associated with C. roseus. Further, it was sensitive enough to amplify direct PCR products obtained from crude DNA template (symptomatic leaf tissues) diluted to 10−3. The authenticity of the PCR-amplified DNA fragment was confirmed after nucleotide sequence determination and restriction fragment length polymorphism pattern. The developed nested PCR based system should facilitate indexing of the phytoplasma in different stages of in vitro regenerated C. roseus plants following

Isolation of Sperm Vesicles from Adult Male Mayflies and Other Insects to Prepare High Molecular Weight Genomic DNA Samples

We describe here a simple and efficient protocol for genomic DNA isolation from adult males of insects: e.g., Ephemeroptera, Odonata, Orthoptera and Dictyoptera. To minimize contamination of external DNA source, the sperm vesicles were isolated from male individuals from which high molecular weight genomic DNA was extracted. According to this protocol, the genomic DNA samples obtained were high quality (intact), and abundant enough for genotyping analyses and molecular cloning. The protocol reported here enables us to process a huge number of individuals at a time with escaping from cross-contamination, and thus it is quite useful for conducting genetic studies at least in some species of insects. The large yield of high molecular weight DNA from single individual may be advantageous for non PCR-based experiments. As a case study of the protocol, partial coding sequences of histone H3 and EF-1alpha genes are determined for some insects with PCR-amplified DNA fragments.

DNA Mutation Detection Using Denaturing High‐Performance Liquid Chromatography (DHPLC)

DHPLC is an efficient method for candidate gene scanning with a high level of automation. Single-base substitutions and insertions or deletions of up to 1.5 kb in PCR amplified DNA fragments can be detected. The method exploits the differential retention of homoduplex and heteroduplex DNA species under conditions of partial thermal denaturation. DHPLC provides a useful platform for high-throughput mutation detection and SNP discovery.

Highly Efficient Yeast-Based in Vivo DNA Cloning of Multiple DNA Fragments and the Simultaneous Construction of Yeast/ Escherichia Coli Shuttle Vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.

Detection of Epstein–Barr virus infection and gene expression in human tumors by microarray analysis

Epstein–Barr virus (EBV) genome-chips are employed to determine the EBV infection rate and to reveal the gene expression patterns of EBV in tumor biopsies. These chips are produced with 71 consecutive PCR-amplified EBV DNA fragments of 1–3 kbp covering the entire EBV genome. The specificity of the EBV-chips is determined by hybridizing the DNA on the chips with biotin-labeled cDNA probes reverse transcribed from the mRNA of P3HR1 cells, which were B-cell infected latently by EBV. Hybridization results revealed only the expression of EBNA1, EBNA2, EBER1 and EBER2 in these cells. On the other hand, EBV lytic genes are expressed after the cells are treated with 12- O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce the EBV lytic cycle. Fourty-four tumor biopsies from different organs are assayed with these chips, which showed many defined and interesting EBV gene expression patterns. This study demonstrates that the EBV-chip is useful for screening infection with EBV in tumors, which may lead to insights into tumorigenesis associated with this virus.

Reevaluation of the Predicted Gene Structure of Dictyostelium Cystatin A3 (cpiC) by Nucleotide Sequence Determination of its cDNA* and its Phylogenetic Position in the Cystatin Superfamily

Cystatins, cysteine protease inhibitors, are widely distributed among eukaryotes. We reevaluated the structure of the gene cpiC, a gene encoding the third identified member of cystatin family (cystatin A3) that was predicted in the genome database of the social amoeba Dictyostelium discoidium (dictyBase) but remained controversial. We determined the sequences of cDNA and PCR-amplified genomic DNA fragment and found a critical error in the registered nucleotide sequence. The corrected cystatin A3 gene has an open reading frame (ORF) without intron sequence interruption and encodes 94 amino acids (aa), in contrast to the previously predicted sequence of either 80, 82 or 118 aa. The cDNA has an unusual internal poly(A) sequence of 31 adenines, which immediately follows the translation termination codon (TAA) located 146 nucleotides upstream of the post-transcriptional polyadenylation site. The amino acid sequence of Dictyostelium cystatin A3 shows a high similarity to those of previously reported Dictyostelium cystatins as well as Family I cystatins of higher eukaryotes.

CDK Phosphorylation Inhibits the DNA-binding and ATP-hydrolysis Activities of the Drosophila Origin Recognition Complex

Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation. Here we show that Drosophila melanogaster ORC (DmORC) is phosphorylated in vivo and is a substrate for Cdks in vitro. Cdk phosphorylation of DmORC subunits DmOrc1p and DmOrc2p inhibits the intrinsic ATPase activity of DmORC without affecting ATP binding to DmOrc1p. Moreover, Cdk phosphorylation inhibits the ATP-dependent DNA-binding activity of DmORC in vitro, thus identifying a novel determinant for DmORC-DNA interaction. DmORC is a substrate for both Cdk2 x cyclin E and Cdk1 x cyclin B in vitro. Such phosphorylation of DmORC by Cdk2 x cyclin E, but not by Cdk1 x cyclin B, requires an "RXL" motif in DmOrc1p. We also identify casein kinase 2 (CK2) as a kinase activity in embryonic extracts targeting DmORC for modification. CK2 phosphorylation does not affect ATP hydrolysis by DmORC but modulates the ATP-dependent DNA-binding activity of DmORC. These results suggest molecular mechanisms by which Cdks may inhibit ORC function as part of re-replication control and show that DmORC activity may be modulated in response to phosphorylation by multiple kinases.

Directional Enrichment of Directly Cloned PCR Products

Reorienting Directional Cloning The cloning of PCR-amplified DNA fragments is frequently the first step in expression vector construction. Commercial kits that exploit the tendency of DNA polymeras...

The ITS2 ribosomal DNA of Anopheles beklemishevi and further remarks on the phylogenetic relationships within the Anopheles maculipennis group of species (Diptera: Culicidae)

Anopheles beklemishevi specimens from Russia were analysed by their ITS2 ribosomal DNA sequence to amend and to specify the phylogenetic tree of the Anopheles maculipennis species complex. Surprisingly, with 638 base pairs, the ITS2 regions of all the 34 An beklemishevi specimens examined were considerably longer than those of all their sibling species. Sequence alignment with GenBank derived sequences of the other siblings was only possible in the beginning (for approx. 335 bp) and at the end (for approx. 150 bp) of the PCR-amplified DNA fragment, whereas in the middle, the An beklemishevi DNA sequence found no counterpart in sequences of the other siblings. Closer analysis of this intermediate part suggests a duplicated insertion of about 140 bp that has undergone subsequent mutational changes. Due to this large putative insertion, computerized phylogenetic analysis by the Bayesian inference method locates An beklemishevi in a closer relationship to the nearctic than to the palaearctic sibling species. However, when only ITS2 regions are compared, that have corresponding sequences in the other siblings, An beklemishevi forms a lineage with the palaearctic species although it is still most remotely related. It is hypothesized that during the evolution An beklemishevi separated first from the common ancestor of the palaearctic species, which had presumably made its way from the Nearctic to the Palaearctic.

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

The Spatial Organization of Lipid Synthesis in the Yeast Saccharomyces cerevisiae Derived from Large Scale Green Fluorescent Protein Tagging and High Resolution Microscopy

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.

Bacillolysin MA, a Novel Bacterial Metalloproteinase That Produces Angiostatin-like Fragments from Plasminogen and Activates Protease Zymogens in the Coagulation and Fibrinolysis Systems

We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.

A Secretory-type Protein, Containing a Pentraxin Domain, Interacts with an A-type K+ Channel

A-type K(+) channels belonging to the Shal subfamily are found in various receptor and neuronal cells. Although their kinetics and cell surface expression are regulated by auxiliary subunits, little is known about the proteins that may interact with Kv4 during development. A yeast two-hybrid screening of a cDNA library made from the sensory epithelium of embryonic chick cochlea revealed a novel association of Kv4.2 with a protein containing a pentraxin domain (PPTX). Sequence analysis shows that PPTX is a member of the long pentraxin family, is 53% identical to mouse PTX3, and has a signal peptide at the N terminus. Studies with chick cochlear tissues reveal that Kv4.2 coprecipitates PPTX and that both proteins are colocalized to the sensory and ganglion cells. A yeast two-hybrid assay demonstrated that the last 22 amino acids of the PPTX C terminus interact with the N terminus of Kv4.2. Chinese hamster ovary cells transfected with recombinant PPTX reveal secretory products in both non-truncated and truncated forms. Among the secreted variants are several blocked by Brefeldin A, suggesting export via a classical pathway. PPTX is soluble in the presence of sodium carbonate, suggesting localization to the cytosolic side of the plasmalemma. Immunohistochemical studies show that Kv4.2 and PPTX colocalize in the region of the plasmalemma of Chinese hamster ovary cells; however, both are locked in the endoplasmic reticulum of COS-7 cells, suggesting that PPTX does not act as a shuttle protein. Reverse transcription-PCR demonstrates that PPTX mRNA is found in tissues that include brain, eye, heart, and blood vessels.

Sustained Transgene Expression in Rat Kidney with Naked Plasmid DNA and PCR-Amplified DNA Fragments

Recently, we developed a kidney-targeted gene transfer technique, in which naked DNA was injected into the renal vein while the renal vein and artery were clamped. Kidney-targeted DNA transfer with only the renal vein clamped is an important modification that may permit less invasive catheter-based gene transfer in future clinical applications. The preparation of PCR-amplified DNA fragments is less time-consuming than that of naked plasmid DNA. We examined rat erythropoietin (Epo) plasmid, pCAGGS-Epo, or PCR-amplified DNA fragment, fCAGGS-Epo, transfer into the rat kidney with only the renal vein clamped. The Epo level peaked at week 3 and then was sustained for 24 weeks, which resulted in significant erythropoiesis. This modified technique, allowing long-term expression of both PCR-amplified DNA fragments and naked plasmid DNA, could potentially be used for catheter-based gene transfer in humans, and could help determine the physiological functions of putative genes.

Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis

Species-specific PCR was used for the identification of nine Fusarium species in pure mycelial culture. A PCR-based method was compared with the whole seed agar plate method and trichothecene analysis for three toxin-producing Fusarium species using 85 grain samples of wheat, barley, oat, corn and rye. A simple SDS-based DNA extraction system followed by potassium acetate precipitation resulted in consistent PCR amplification of DNA fragments from cultures and grain samples. The species-specific PCR assays correctly identified pure cultures of Fusarium avenaceum ssp. avenaceum (9 isolates), Fusarium acuminatum ssp. acuminatum (12 isolates), Fusarium crookwellense (7 isolates), Fusarium culmorum (12 isolates), Fusarium equiseti (11 isolates), Fusarium graminearum (77 isolates), Fusarium poae (10 isolates), Fusarium pseudograminearum (23 isolates), and Fusarium sporotrichioides (10 isolates). Multiplex PCR was developed for the simultaneous detection of F. culmorum, F. graminearum and F. sporotrichioides, the three most important trichothecene producing species in Canada. In grain samples, results of PCR assays for these same three species related well with whole seed agar plate method results and determination of Fusarium trichothecenes. The PCR assay described in this study can be used for routine detection and identification of Fusarium spp. in Canada.

Banana contains a diverse array of endogenous badnaviruses

Banana streak disease is caused by several distinct badnavirus species, one of which is Banana streak Obino l'Ewai virus. Banana streak Obino l'Ewai virus has severely hindered international banana (Musa spp.) breeding programmes, as new hybrids are frequently infected with this virus, curtailing any further exploitation. This infection is thought to arise from viral DNA integrated in the nuclear genome of Musa balbisiana (B genome), one of the wild species contributing to many of the banana cultivars currently grown. In order to determine whether the DNA of other badnavirus species is integrated in the Musa genome, PCR-amplified DNA fragments from Musa acuminata, M. balbisiana and Musa schizocarpa, as well as cultivars 'Obino l'Ewai' and 'Klue Tiparot', were cloned. In total, 103 clones were sequenced and all had similarity to open reading frame III in the badnavirus genome, although there was remarkable variation, with 36 distinct sequences being recognized with less than 85 % nucleotide identity to each other. There was no commonality in the sequences amplified from M. acuminata and M. balbisiana, suggesting that integration occurred following the separation of these species. Analysis of rates of non-synonymous and synonymous substitution suggested that the integrated sequences evolved under a high degree of selective constraint as might be expected for a living badnavirus, and that each distinct sequence resulted from an independent integration event.