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PubMed2003

Creating precise GFP fusions in plasmids using yeast homologous recombination.

Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.

Animals↗
PubMed1994

The formation of one-G deletions as a consequence of single-oxygen-induced DNA damage.

Single-stranded M13mp10 DNA containing a 144-bp mutational target sequence in the lacZ alpha gene was treated with singlet oxygen (1O2) generated by thermodissociation of the endoperoxide of 3,3'-(1,4-naphthalene-1,4-diyl)dipropionate (NDPO2). After transfection to non-SOS-induced E. coli cells, 32 mutants preselected for a mutation in the 144-bp target were collected and analyzed by DNA sequencing. One-G deletions represented the predominant type of mutation accounting for 50% of the mutations analyzed. The remaining part appeared to consist of base substitutions, i.e. G-->T transversions (34%), C-->T transitions (12.5%) and one T-->C transition (3%). Sixty percent of the mutations were found in two major mutational hotspots. We conclude that the predominant one-G deletions are due to a guanine reaction product which might be specific for 1O2.

Bacteriophage M13↗
PubMed2006

DrugBank: a comprehensive resource for in silico drug discovery and exploration.

DrugBank is a unique bioinformatics/cheminformatics resource that combines detailed drug (i.e. chemical) data with comprehensive drug target (i.e. protein) information. The database contains >4100 drug entries including >800 FDA approved small molecule and biotech drugs as well as >3200 experimental drugs. Additionally, >14,000 protein or drug target sequences are linked to these drug entries. Each DrugCard entry contains >80 data fields with half of the information being devoted to drug/chemical data and the other half devoted to drug target or protein data. Many data fields are hyperlinked to other databases (KEGG, PubChem, ChEBI, PDB, Swiss-Prot and GenBank) and a variety of structure viewing applets. The database is fully searchable supporting extensive text, sequence, chemical structure and relational query searches. Potential applications of DrugBank include in silico drug target discovery, drug design, drug docking or screening, drug metabolism prediction, drug interaction prediction and general pharmaceutical education. DrugBank is available at http://redpoll.pharmacy.ualberta.ca/drugbank/.

Computational Biology↗
PubMed1993

Evidence for an interaction between the CYP1(HAP1) activator and a cellular factor during heme-dependent transcriptional regulation in the yeast Saccharomyces cerevisiae.

Previously, it was shown that the CYP1(HAP1) gene product mediates the transcription of several oxygen-regulated genes through a metabolic co-effector, heme, in the yeast Saccharomyces cerevisiae. This study investigates the overproduction of the CYP1 protein when the CYP1(HAP1) gene is placed under the control of the GAL10-CYC1 hybrid promoter (either at the locus of the CYP1(HAP1) gene or cloned in a high-copy-number plasmid). In these conditions, the CYP1 protein is detected by Western blot analysis and has a molecular mass in agreement with the open reading frame sequence. Band-shift experiments show that the CYP1(HAP1) protein is able to interact specifically with its target sequences in vitro without addition of hemin, and forms a large complex with one or several unidentified factors denoted as X. Addition of hemin allows the formation of a new complex which has a lower molecular mass. The internal deletion of the seven repeated amino acid sequences containing the KCPVDH motif in the CYP1(HAP1) protein modifies the heme responsiveness phenomenon observed in vitro in the band-shift experiments and in vivo in the transcription of the CYB2, CYC1, CYP3(CYC7) and ERG11 genes. On the basis of these data, we propose a new model for heme-induced activation of the CYP1 protein.

Amino Acid Sequence↗
PubMed1996

Reciprocal regulation of delta 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants.

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745 bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the delta 1-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1992

DNA binding activity is required for EBNA 1-dependent transcriptional activation and DNA replication.

Epstein-Barr virus nuclear antigen 1 (EBNA 1) has been shown to be a sequence-specific DNA binding protein that is required for the replication of episomal elements carrying the viral origin of DNA replication, oriP, as well as for the activation of a specific transcriptional enhancer. We have constructed and analyzed a series of deletion and nonsense mutants in a cloned copy of the EBNA 1 gene and have tested mutant peptides for the ability (a) to bind to a synthetic oligonucleotide containing a consensus EBNA 1 binding site, (b) to activate the EBNA 1-specific enhancer, and (c) to drive replication of an oriP-bearing plasmid in a transient replication assay. The presence of a DNA binding domain in the carboxy-terminal third of the protein was confirmed. Interestingly, neither the acidic tail nor the Gly-Ala copolymer of EBNA 1 contributes significantly to binding. In addition to sequences in the carboxy-terminal portion of the protein, our data indicate that sequences in the amino-terminal portion of the polypeptide affect the binding of EBNA 1 to its target sequence. Further, we show that EBNA 1 binds to its recognition sequence as a dimer. Results of transient expression assays indicate that the ability of EBNA 1 species to activate the transcriptional enhancer and to drive the replication of oriP plasmids is directly dependent on the ability of the polypeptides to bind to the EBNA 1 consensus binding sequence.

Antigens, Viral↗
PubMed2006

A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons.

We have developed a new method for the detection of nucleic acid hybridization, based on a simple latex agglutination test that can be evaluated by the unaided eye. Nucleic acid, e.g., a polymerase chain reaction (PCR) product, is denatured and incubated with polystyrene beads carrying covalently bound complementary oligonucleotide sequences. Hybridization of the nucleic acids leads to aggregation of the latex particles, thereby verifying the presence of target sequence. The test is performed at room temperature, and results are available within 10 min. As a proof of principle, the hybridization/latex agglutination assay was applied to the detection of purified PCR fragments either specific for Salmonella spp. or a synthetic sequence, and to the detection of Salmonella enterica in artificially contaminated chicken samples. A few nanograms of purified PCR fragments were detectable. In artificially contaminated chicken samples, 3 colony-forming units (cfu)/25 g were detected in one of three replicates, and 30 cfu/25 g were detected in both of two replicates when samples for PCR were taken directly from primary enrichment, demonstrating the practical applicability of this test system. Even multiplex detection might be achievable. This novel kind of assay could be useful for a range of applications where hybridization of nucleic acids, e.g., PCR fragments, is to be detected.

Animals↗
PubMed2001

The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 is an alpha-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Photosynthesis may be photoheterotrophic using organic compounds as both a carbon and a reducing source, or photoautotrophic using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. The structural components of this metabolically diverse organism and their modes of integrated regulation are encoded by a genome of approximately 4.5 Mb in size. The genome comprises two chromosomes CI and CII (2.9 and 0.9 Mb, respectively) and five other replicons. Sequencing of the genome has been carried out by two groups, the Joint Genome Institute, which carried out shotgun-sequencing of the entire genome and The University of Texas-Houston Medical School, which carried out a targeted sequencing strategy of CII. Here we describe our current understanding of the genome when data from both of these groups are combined. Previous work had suggested that the two chromosomes are equal partners sharing responsibilities for fundamental cellular processes. This view has been reinforced by our preliminary analysis of the virtually completed genome sequence. We also have some evidence to suggest that two of the plasmids, pRS241a and pRS241b encode chromosomal type functions and their role may be more than that of accessory elements, perhaps representing replicons in a transition state.

Journal Article↗
PubMedDate not supplied

siRNA, miRNA and HIV: promises and challenges.

Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).

HIV↗
PubMed2000

Transcription and replication of mitochondrial DNA.

The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content. Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophage-like, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A. One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript. In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus. Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects. Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.

Animals↗
PubMed1991

A directed DNA sequencing strategy based upon Tn3 transposon mutagenesis: application to the ADE1 locus on Saccharomyces cerevisiae chromosome I.

We have developed a directed DNA sequencing strategy based upon the Escherichia coli transposon Tn3. This transposon displays little sequence specificity for transposition and is thus well suited to this task. Both mini-Tn3 transposons and sequencing vectors bearing the phage f1 single stranded origin of replication have been constructed. Upon mutagenesis of a target sequence, a population is produced in which each clone has two f1 origins of replication, one of which is at a variable position depending upon the transposon insertion site. When helper phage is added to the mutagenised population, the two f1 origins present on each clone are nicked, dividing the packaged strand into two segments, each of which is packaged into a separate phage particle. One of these segments contains no resistance markers and is lost, whilst the other is recovered as a deleted clone with a single chimeric f1 origin. A unidirectionally, variably-deleted set of sequencing clones is produced, and appropriately sized clones are sequenced using a primer complimentary to the transposon end. In addition to being inexpensive, the method does not require the same degree of technical expertise needed for many in vitro, enzymatically based methods. The strategy has been used to determine 2.6 kilobases of nucleotide sequence in the Saccharomyces cerevisiae ADE 1 locus.

Amino Acid Sequence↗
PubMed2001

Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems.

Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.

Amino Acid Sequence↗
PubMed1990

Intramolecular transposition by a synthetic IS50 (Tn5) derivative.

We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences.

Base Sequence↗
PubMed1992

Development of a polymerase chain reaction-based diagnosis of Trichomonas vaginalis.

We developed a polymerase chain reaction (PCR)-based test for detecting the protozoan parasite Trichomonas vaginalis. Genomic libraries were constructed from two independent clinical isolates of T. vaginalis. From these libraries, 12 genomic clones were purified, sequenced, and then screened for uniqueness by computer-assisted sequence comparisons. PCR reactions were performed to evaluate eight PCR-primer pairs, including a primer pair that targeted the T. vaginalis ferredoxin gene. All eight primer pairs yielded PCR products of the expected sizes. However, six of the primer pairs amplified their respective target sequences in limited numbers of clinical T. vaginalis isolates, suggesting the presence of significant genomic variability among isolates. An exception was a primer pair, termed TVA5-TVA6, that amplified a 102-bp genomic sequence, termed A6p, in all of 24 clinical isolates. The A6p sequence was not detected by PCR in human DNA or in a wide variety of flagellates, ciliates, or bacteria tested. The A6p sequence appears highly selective for a broad range of T. vaginalis isolates and holds promise for PCR-based diagnosis of the parasite.

Animals↗
PubMed1997

Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification.

Multiplex strand displacement amplification (mSDA) is capable of amplifying three distinct DNA sequences simultaneously. These include sequences present in most genera of mycobacteria, a sequence specific for Mycobacterium tuberculosis, and an internal control. mSDA was used to detect the presence of these target sequences in 154 (72 positive, 76 negative, and 6 failed) clinical specimens cultured in the mycobacterial growth indicator tube (MGIT) system. A wide variety of specimen types were processed and cultured. Once these cultures were deemed positive by MGIT fluorescence or were deemed negative after 8 weeks of incubation, MGIT culture aliquots were processed for mSDA analyses. A chemiluminescent microwell assay was used to detect the amplified products. The procedure was relatively simple and took less than 6 h to complete. The sensitivity of mSDA for detecting acid-fast bacilli was 96.4% compared to that of MGIT culture. Sensitivity and specificity were 97.2 and 96.1%, respectively, when all clinical criteria were considered. mSDA was shown to be a rapid and effective method for confirming the presence of M. tuberculosis and other mycobacteria in positive MGIT cultures.

Bacterial Typing Techniques↗
PubMed1986

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor.

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

Adenoviruses, Human↗
PubMed2006

DNPTrapper: an assembly editing tool for finishing and analysis of complex repeat regions.

BACKGROUND: Many genome projects are left unfinished due to complex, repeated regions. Finishing is the most time consuming step in sequencing and current finishing tools are not designed with particular attention to the repeat problem. RESULTS: We have developed DNPTrapper, a shotgun sequence finishing tool, specifically designed to address the problems posed by the presence of repeated regions in the target sequence. The program detects and visualizes single base differences between nearly identical repeat copies, and offers the overview and flexibility needed to rapidly resolve complex regions within a working session. The use of a database allows large amounts of data to be stored and handled, and allows viewing of mammalian size genomes. The program is available under an Open Source license. CONCLUSION: With DNPTrapper, it is possible to separate repeated regions that previously were considered impossible to resolve, and finishing tasks that previously took days or weeks can be resolved within hours or even minutes.

Algorithms↗
PubMed2000

Identification and characterization of a cDNA clone encoding the heat shock protein (Hsp60) from the biting midge, Culicoides variipennis sonorensis Wirth and Jones.

A cDNA expression library constructed from Culicoides variipennis sonorensis was screened using an antibody specific for Hsp60 of Heliothis virescens. A single clone encoding the complete heat shock protein (Hsp60) of C. variipennis was identified and its 2400-bp insert was sequenced. The encoded 62-kDa protein contains 581 amino acids and includes a 26-amino acid putative mitochondrial targeting sequence at its N terminus and a GGM motif at its carboxyl terminus. Deduced amino acid sequences are highly similar (67-78%) to Hsp60 of other species, including the fruit fly, the house mouse, the Norwegian rat, the Chinese hamster, the human, a nematode, and the tobacco budworm moth. This is the initial isolation of a coding sequence for a stress-induced protein in C. variipennis.

Amino Acid Sequence↗