Isolation Of DNA Sequence Research Articles

Chloroquine resistance in Plasmodium falciparum and polymorphism of the CG2 gene.

A distinct genotype (designated Dd2-type profile) consisting of 12 point mutations and 3 repetitive regions of the CG2 gene, a candidate gene for chloroquine resistance, has been associated with in vitro resistance in laboratory-adapted strains of Plasmodium falciparum. The DNA sequence of clinical isolates, characterized by in vitro and in vivo tests, was analyzed to evaluate whether the genotype corresponds to the phenotype in naturally occurring parasites. Eight of 11 chloroquine-resistant isolates had the Dd2 genotype. One resistant isolate (by in vitro assay) with a sensitive CG2 genotype was sensitive in vivo. Two resistant isolates and 6 sensitive isolates were multiple infections with mixed alleles. No typical CG2 genotype was found corresponding to the chloroquine-sensitive isolates. These results suggest a strong association between the drug-resistant and CG2 genotypes and support the hypothesis that the CG2 gene may be implicated in chloroquine resistance.

A simplified subtractive hybridization protocol used to isolate DNA sequences specific to Xylella fastidiosa.

A simplified protocol of subtractive hybridization based on the technique of L. M. Kunkel, A. P. Monaco, W. Middlesworth, H. D. Ochs & S. A. Latt (1985, Proc Natl Acad Sci USA, 82, 4778-4782) was used to obtain DNA sequences specific to Xylella fastidiosa isolated from diseased citrus plants. As a driver, DNA extracted from bacteria showing different degrees of relatedness was used: Xy. fastidiosa 788 isolated from another host (plum), Xanthomonas campestris pv. campestris and Burkholderia gladioli strains. A DNA fragment, f14, showing no hybridization to the driver DNA, was used as a probe specific to Xy. fastidiosa from citrus and oleander. This fragment was sequenced and the predicted protein showed 40% similarity to the central region of flagellin of Escherichia coli serotypes H1 and H12. A pair of internal primers (f14-1 and f14-2) was designed for amplification of Xy. fastidiosa DNA. These primers detected Xy. fastidiosa strains isolated from citrus and oleander and yielded an amplification product of about 600 bp. They were also able to detect the bacteria in extracts from citrus plants with or without symptoms of disease. No amplification reaction was obtained using DNA extracted from other species and pathovars of Xanthomonas, Pseudomonas cichorii, Erwinia carotovora, Agrobacterium tumefaciens and phytopathogens of citrus (Xanthomonas axonopodis pv. citri) and coffee (Burkholderia andropogonis, P. cichorii, Pseudomonas syringae pv. garcae). The isolation of a DNA fragment specific to Xy. fastidiosa from citrus showed that the simplified protocol of subtractive hybridization used in this work is potentially applicable to other micro-organisms.

Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences

Our laboratories recently completed SELEX experiments to isolate DNA sequences that most-strongly favor or disfavor nucleosome formation and positioning, from the entire mouse genome or from even more diverse pools of chemically synthetic random sequence DNA. Here we directly compare these selected natural and non-natural sequences. We find that the strongest natural positioning sequences have affinities for histone binding and nucleosome formation that are sixfold or more lower than those possessed by many of the selected non-natural sequences. We conclude that even the highest-affinity sequence regions of eukaryotic genomes are not evolved for the highest affinity or nucleosome positioning power. Fourier transform calculations on the selected natural sequences reveal a special significance for nucleosome positioning of a motif consisting of ∼10 bp periodic placement of TA dinucleotide steps. Contributions to histone binding and nucleosome formation from periodic TA steps are more significant than those from other periodic steps such as AA (=TT), CC (=GG) and more important than those from the other YR steps (CA (=TG) and CG), which are reported to have greater conformational flexibility in protein-DNA complexes even than TA. We report the development of improved procedures for measuring the free energies of even stronger positioning sequences that may be isolated in the future, and show that when the favorable free energy of histone-DNA interactions becomes sufficiently large, measurements based on the widely used exchange method become unreliable.

Identification of DNA binding sites for the v-erbA oncoprotein, the viral homolog to thyroid hormone receptor alpha

The v-erbA oncogene protein, P75 gag-v-erbA, is a mutant form of the thyroid hormone receptor α (TR α) which has sustained mutations both in the ligand binding and DNA binding domains. The oncoprotein has therefore lost its ability to bind ligand, and its heterodimerization with the retinoid-X receptor (RXR) is impaired. Here, we have investigated the effects of the mutations in the DNA binding domain. By applying a PCR-based screening assay we isolated DNA sequences to which P75 gag-v-erbA binds as a heterodimer with RXR, and characterized these with regard to their nucleotide sequence and ability to associate with RXR/P75 gag-v-erbA heterodimers in vitro and in vivo. In the PCR selection assay the heterodimer exhibited a preference for direct repeats with a 3′ half-site sequence AGGTCG and spacers of four or five nucleotides separating the two half-sites. These DNA binding data were confirmed by gel retardation assays with synthetic oligonucleotides as well as by transfection experiments using dominantly active VP16 fusion proteins with P75 gag-v-erbA and TR α. The comparison between RXR/P75 gag-v-erbA and RXR/TR α heterodimers demonstrated that although their DNA binding properties are very similar, however, a relaxed specificity of P75 gag-v-erbA for the spacer length may allow it to interfere with more hormone signalling pathways than only that of thyroid hormone.

A simple PCR-based method for scoring the ph1b deletion in wheat

The amplified fragment length polymorphism (AFLP) technique was used to isolate DNA sequences present in the euploid wheat Chinese Spring but not in the Chinese Spring ph1b mutant (which has a deletion of the Ph1 gene, a suppressor of homoeologous chromosome pairing). The polymorphic DNA fragments identified by AFLP were then cloned, sequenced, and used to design two primer pairs. These primers were used in a PCR-based assay to specifically amplify products from the Chinese Spring euploid but not from the ph1b mutant. This PCR assay can be carried out from extracted genomic DNA or directly from alkaline-treated wheat leaves, and the reaction products can be scored on a plus-minus basis, making the screening amenable to automation. The reliability of the assay was tested using a F1-derived doubled-haploid population of 55 lines which segregate for the ph1b deletion. This PCR-screening technique is less time and labour consuming, and more accurate and reliable, than cytologically based conventional methods.

A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences.

A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.

A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences

A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.

Simian T-Lymphotropic Virus Type 1 (STLV-1) Infection in Wild Yellow Baboons (Papio hamadryas cynocephalus) from Mikumi National Park, Tanzania

Serum and peripheral blood leukocytes from wild yellow baboons (Papio hamadryas cynocephalus) were tested for the presence of STLV-1-specific antibodies and proviral DNA. Fourteen of 30 sera tested positive by radioimmunoprecipitation assay (RIPA) with HTLV-1. Among 36 DNA samples tested by PCR 15 were positive by double nested PCR for a fragment of the STLV-1envgene, the most sensitive assay among PCR tests employed. Of 30 animals that were tested both serologically and by PCR in only 1 case were the results discordant (PCR-positive, antibody-negative). The DNA sequences fromenv(378 bp),pol(212 bp), andLTR(705 bp) were determined for 5, 5, and 2 Mikumi STLV-1 isolates, respectively. The DNA sequences of Mikumi STLV-1 isolates were virtually identical and phylogenetic analysis revealed that they were clearly distinct from previously published baboon STLV-1 sequences, including those STLV-1 isolates presumed to be from yellow baboons. The results of this study suggest that reliable placement of individual STLV-1 within the PTLV-1 phylogeny requires genomic sequences of STLV-1 isolates from wild animals whose taxonomic identity and geographical origin are firmly established and that the LTR is the genomic region of STLV-1 which is the most informative for cladistic analysis of these viruses.

Human Herpesvirus 8 Sequences Are Not Detected in Epithelial Tumors From Patients Receiving Transplants

Recently, Chang et al 1 isolated DNA sequences from a new herpesvirus, human herpesvirus 8 (HHV-8), from Kaposi sarcoma (KS) specimens. Using polymerase chain reaction (PCR) with the KS330 233 primers, Rady et al 2 detected 233—base pair products in various epithelial skin tumors from 4 immunosuppressed patients. Boshoff et al 3 did not confirm these data in 37 squamous cell carcinoma specimens from 25 immunosuppressed and 6 immunocompetent individuals using nested PCR with 2 sets of primers derived from the ORF26 gene and the conserved HHV-8 major capsid protein gene, ORF25 . Therefore, further data are needed concerning these controversial findings on the presence of HHV-8 in lesions other than KS. We have investigated the presence of HHV-8 sequences in 12 squamous cell carcinoma specimens, 6 basal cell carcinoma specimens, and 2 actinic keratosis specimens from 17 different immunosuppressed patients receiving transplants. The DNA was extracted from paraffin wax—embedded tissues,

Primary structure of a ferredoxin-like iron-sulfur subunit of complex I from Neurospora crassa

We have isolated cDNA clones encoding an iron-sulfur polypeptide subunit of the mitochondrial complex I of Neurospora crassa. The fungal cDNA library was screened by hybridisation with an heterologous probe from Paracoccus denitrificans. The DNA sequence of relevant isolates was determined and revealed an open reading frame encoding a precursor protein of 219 amino acid residues. The gene product is a ferredoxin-like protein that contains two cysteine-rich motives that may each bind a tetranuclear iron-sulfur cluster. The primary structure of the protein is highly homologous to the 23 kDa iron-sulfur subunit of complex I from bovine and P. denitrificans. Interestingly, an alanine residue within the second cluster-binding motif, which is conserved in complex I but replaced by tyrosine in similar chloroplast genes, is substituted for serine in N. crassa.

Development of a method for the identification, using the polymerase chain reaction, of Aspergillus fumigatus isolated from ostriches.

To develop a method for identifying DNA of Aspergillus fumigatus from ostriches, using the polymerase chain reaction (PCR). A fumigatus is the principal causative agent of avian aspergillosis. A biochemical trial. Twelve Aspergillus fumigatus isolates and three other Aspergillus species. PCR primers that were based on the sequence of the alkaline protease gene from human isolates of A fumigatus were used. We successfully tested the method on ostrich isolates from five states and showed that the test is specific for A fumigatus. In most cases the DNA sequence of A fumigatus isolates from ostriches is similar to that of human isolates. DNA sequences vary significantly among A fumigatus isolates, including those from affected ostriches in the same flock. The genetic variation may be used to trace aspergillus infections in ostrich flocks and determine if the disease is transmitted by contact with infected birds.

Coincidence cloning. Taking the coincidences out of genome analysis.

The term "coincidence cloning" encompasses a wide range of methodologies, the aim of which is to isolate DNA sequences which occur in both of two input DNA sources. The nature of these input DNAs may be genomic or cDNA, cloned or uncloned, and as such the far reaching applicability of the techniques can be imagined. If the input DNAs are genomic then the product will be enriched for useful markers co-occurring between the two. If the input DNAs comprise one genomic resource and one cDNA resource the product will contain genes mapping to that particular genomic region. In this review a comparative description of the range of coincidence cloning methods is given, together with a discussion of their applications. Finally, consideration is given to the general limitations of these techniques.

Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria.

To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.

The Isolation and Characterization of the Human Cytochrome b5 Gene

From a series of lambda and cosmid libraries, we isolated DNA sequences corresponding to the complete coding region for the human cytochrome b5 (CYB5) gene. The overall gene organization was the same as the bovine and rabbit CYB5 genes. One cosmid clone containing exon I plus the 5′ flanking region was extensively characterized. From this clone we obtained 2000 bp of 5′ flanking sequence, containing several distinctive GC rich regions, potential trans-acting factor binding sites and a 74 bp direct repeat. A series of deletion constructs were made in the pGL2 luciferase vector and then used to transfect HepG2 and K562 cells. The data obtained suggest the presence of two promoters and one silencer region in the analyzed 5′ sequence.

Use of Continuous Culture for the Selection of Plasmids with Improved Segregational Stability

In this report a method that enables the selection of stable plasmid variants and the isolation of DNA sequences that improve plasmid maintenance is described. The method is based on the principle that in populations of cells carrying derivatives of a plasmid that differ only in the level of segregational stability, when grown in a chemoslat under conditions with selective pressure on the plasmid, cells that carry more stable plasmid variants will be enriched. We developed the system for Lactococcus lactis using segregationally unstable derivatives of the gram-positive theta plasmid pAMβ1 as selection vectors. The results showed that the method is suitable for the enrichment, and subsequent purification, of three classes of plasmids with improved maintenance properties. The first class involved mutations in the pAMβ1-derived selection plasmid. These mutations resulted in increased copy numbers, thereby rendering the plasmid segregationally more stable. The other two classes were based on the insertion of additional, stability promoting. sequences in the selection plasmids. We showed that these sequences can constitute either replication functions derived from another plasmid or functions directly involved in plasmid maintenance. The site-specific resolution function of pAMβ1 was used as an example of the latter functions. We anticipate that the method described should have general applicability for the development of stable host-vector systems in bacteria.

DNA Probes Can Be Used To Discriminate between Tephritid Species at all Stages of the Life Cycle (Diptera: Tephritidae)

We isolated DNA sequences from the genomes of three Tephritid species. These can be used as probes to make rapid and reliable species identifications using material from any stage of the life cycle. The three Tephritid species are the Mediterranean fruit fly, Ceratitis capitata (Weidemann); the oriental fruit fly, Bactrocera dorsalis (Hendel), and the melon fly, Bactrocera cucurbitae (Coquillett). These isolated DNA sequences can be used to probe specimen material such as single eggs, larvae, or adult body parts on dot blots or squash blots to determine rapidly which species are present when infestations are found. The actual species identifications are made either on the basis of a direct positive result with a particular probe or by the process of elimination from multiple probings. The availability of species-specific DNA probes for use on minimal amounts of material from any stage of the life cycle or body part can facilitate greatly the identification of specimens which are otherwise indistinguishable or unsuitable for classification on the basis of morphological criteria. This technique also allows material to be analyzed immediately, which obviates the need for rearing and handling of material collected at immature stages. Although these probes have been developed for the three Tephritid species described here, the methods used are applicable to any group of organisms.

Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.

Application of subtraction hybridization for the development of a Rhizobium leguminosarum biovar phaseoli and Rhizobium tropici group-specific DNA probe

A combined subtraction hybridization and polymerase chain reaction/amplification technique was used to develop a DNA probe which was specific for the Rhizobium leguminosarum biovar phaseoli and the Rhizobium tropici group. Total genomic DNA preparations from Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar trifolii, Rhizobium sp., Agrobacterium tumefaciens, Rhizobium fredii, Bradyrhizobium japonicum, Bradyrhizobium ssp. and Rhizobium meliloti were pooled and used as subtracter DNA against total genomic DNA from the Rhizobium leguminosarum biovar phaseolo strain KIM5s. Only one round of subtraction hybridization at 65°C was necessary to remove all cross-hybridizing sequences. Dot blot hybridizations with total genomic DNA of the eight subtracter organisms and 29 bacteria of different groups confirmed the high specificity of the isolated DNA sequences. Dot blot hybridizations and total genomic DNA from ten different R. Leguminosarum biovar phaseoli and R. tropici strains resulted in strong hybridization signals for all strains tested. The DNA probe for the R. tropici and R. leguminosarum biovar phaseoli group was used for dot blot hybridization with DNA extracts from three tropical and one boreal soil. When correlated with data from Most Probable Number analyses the probe was capable of detecting as low as 3 × 104 homologous indigenous rhizobia per g soil. The technique offers great benefits for the development of DNA probes for monitoring bacterial populations in environmental samples.

Application of subtraction hybridization for the development of a Rhizobium leguminosarum biovar phaseoli and Rhizobium tropici group-specific DNA probe

A combined subtraction hybridization and polymerase chain reaction/amplification technique was used to develop a DNA probe which was specific for the Rhizobium leguminosarum biovar phaseoli and the Rhizobium tropici group. Total genomic DNA preparations from Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar trifolii, Rhizobium sp., Agrobacterium tumefaciens, Rhizobium fredii, Bradyrhizobium japonicum, Bradyrhizobium ssp. and Rhizobium meliloti were pooled and used as subtracter DNA against total genomic DNA from the Rhizobium leguminosarum biovar phaseolo strain KIM5s. Only one round of subtraction hybridization at 65°C was necessary to remove all cross-hybridizing sequences. Dot blot hybridizations with total genomic DNA of the eight subtracter organisms and 29 bacteria of different groups confirmed the high specificity of the isolated DNA sequences. Dot blot hybridizations and total genomic DNA from ten different R. Leguminosarum biovar phaseoli and R. tropici strains resulted in strong hybridization signals for all strains tested. The DNA probe for the R. tropici and R. leguminosarum biovar phaseoli group was used for dot blot hybridization with DNA extracts from three tropical and one boreal soil. When correlated with data from Most Probable Number analyses the probe was capable of detecting as low as 3 × 104 homologous indigenous rhizobia per g soil. The technique offers great benefits for the development of DNA probes for monitoring bacterial populations in environmental samples.

Analysis of Repetitive DNA Sequences from Potato Cyst Nematodes and Their Use as Diagnostic Probes

The potato cyst nematode species Globodera rostochiensis and G. pallida pathotype Pa2/Pa3, as well as G. pallida pathotype Pal, can be distinguished from each other by restriction fragment length polymorphisms using DNA probes, which are present at low frequencies in the nematode genome. By differential screening of nematode DNA we have isolated DNA sequences that are highly abundant in G. rostochiensis, G. pallida, or the G. pallida Pal pathotype (...)