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Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis.

We describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of DNA-binding proteins within large DNA molecules. Using this approach, we have mapped E. coli IHF (Integration Host Factor) binding sites within phage Lambda (48 kb) and phage Mu (39 kb) DNA. We are also able to visualize IHF binding sites in E. coli chromosomal DNA (4,700 kb). We present an extension of this technique using direct amplification by PCR of the isolated restriction fragments, which should permit the cloning of a collection of recognition sequences for DNA binding proteins in complex genomes.

Bacterial Proteins↗

Isolation and characterization of novel giant Stenotrophomonas maltophilia phage phiSMA5.

Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated phiSMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The phiSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, phiKZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to phiSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, phiSMA5 appears to be the first reported.

Amino Acid Sequence↗

Expansion of the BioCyc collection of pathway/genome databases to 160 genomes.

The BioCyc database collection is a set of 160 pathway/genome databases (PGDBs) for most eukaryotic and prokaryotic species whose genomes have been completely sequenced to date. Each PGDB in the BioCyc collection describes the genome and predicted metabolic network of a single organism, inferred from the MetaCyc database, which is a reference source on metabolic pathways from multiple organisms. In addition, each bacterial PGDB includes predicted operons for the corresponding species. The BioCyc collection provides a unique resource for computational systems biology, namely global and comparative analyses of genomes and metabolic networks, and a supplement to the BioCyc resource of curated PGDBs. The Omics viewer available through the BioCyc website allows scientists to visualize combinations of gene expression, proteomics and metabolomics data on the metabolic maps of these organisms. This paper discusses the computational methodology by which the BioCyc collection has been expanded, and presents an aggregate analysis of the collection that includes the range of number of pathways present in these organisms, and the most frequently observed pathways. We seek scientists to adopt and curate individual PGDBs within the BioCyc collection. Only by harnessing the expertise of many scientists we can hope to produce biological databases, which accurately reflect the depth and breadth of knowledge that the biomedical research community is producing.

Animals↗

T7 single strand DNA binding protein but not T7 helicase is required for DNA double strand break repair.

An in vitro system based on Escherichia coli infected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome. Double strand breaks were placed in a unique XhoI site located approximately 17% from the left end of the T7 genome. In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage. In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed. Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site. In the present study, it was found that extracts prepared from uninfected E. coli were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism. Extracts prepared from an E. coli recA mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the E. coli recombinase is not essential to the recombinational events required for double strand break repair. In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein. Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect. This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7.

Bacteriophage T7↗

The Mouse Genome Database (MGD): integrating biology with the genome.

The Mouse Genome Database (MGD) is one component of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a community database resource for the laboratory mouse. MGD strives to provide a comprehensive knowledgebase about the mouse with experiments and data annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genetic, genotype (sequence) and phenotype information including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships between genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent developments in MGD discussed here include an extensive integration of the mouse sequence data and substantial revisions in the presentation, query and visualization of sequence data.

Animals↗

The atomic force microscope as a new microdissecting tool for the generation of genetic probes.

The atomic force microscope (AFM) can be used to visualize and to manipulate biological material with relative case and high resolution. This study was carried out to investigate whether probe sets, specific for subregions of the human genome and useful for the painting of chromosome bands, can be established by PCR amplification of AFM-dissected chromosome regions. Compared to standard microdissection techniques, the AFM can be used with much higher precision for the dissection of the region of interest and subsequent nanoextraction of DNA material. After scanning the area of interest in noncontact mode AFM, chromosome bands were cut by the AFM tip at high force. The genetic material of a single cut attached itself to the tip and was extracted and amplified using degenerate oligonucleotide-primed-PCR. Subsequent to hapten labeling, fluorescence in situ hybridization was performed and chromosome band-specific probes were visualized by standard fluorescence microscopy.

Chromosomes, Human, Pair 2↗

Analysis of nucleotide distribution in the genome of Streptomyces coelicolor A3(2) using the Z curve method.

The nucleotide distribution of all 33,527 open reading frames (ORFs) (> or =300 bp) in the genome of Streptomyces coelicolor A3(2) has been analyzed using the Z curve method. Each ORF is mapped onto a point in a 9-dimensional space. To visualize the distribution of mapping points, the points are projected onto the principal plane based on principal component analysis. Consequently, the distribution pattern of the 33,527 points in the principal plane shows a flower-like shape, in which there are seven distinct regions. In addition to the central region, there are six petal-like regions around the center, one of which corresponds to 7172 coding sequences. The central region and the remaining five petal-like regions correspond to the intergenic sequences and out-of-frame non-coding ORFs, respectively. It is shown that selective pressure produces a remarkable bias of the G+C content among three codon positions, resulting in the interesting phenomenon observed. A similar phenomenon is also observed for other bacterial genomes with high genomic G+C content, such as Pseudomonas aeruginosa PA01 (G+C = 66.6%). However, for the genomes of Bacillus subtilis (G+C = 43.5%) and Clostridium perfringens (G+C = 28.6%), no similar phenomenon was observed. The finding presented here may be useful to improve the gene-finding algorithms for genomes with high G+C content. A set of supplementary materials including the plots displaying the base distribution patterns of ORFs in 12 prokaryotes is provided on the website http://tubic.tju.edu.cn/highGC/.

Genome, Bacterial↗

Visualization of molecular activities inside living cells with fluorescent labels.

The major task of modern cell biology is to identify the function and relation of the many different gene products, discovered by genomics and proteomics approaches, in the context of the living cell. To achieve this goal, an increasing toolbox of custom-designed biosensors based on fluorescent labels is available to study the molecular activities of the cellular machinery. An overview of the current status of the young field of molecular-cellular physiology is presented that includes the application of fluorescent labels in the design of biosensors and the major detection schemes used to extract their sensing information. In particular, the use of the photophysical phenomenon of Förster resonance energy transfer (FRET) as a powerful indicator of cellular biochemical events is discussed. In addition, we will point out the challenges and directions of the field and project the short-term future for the application of fluorescence-based biosensors in biology.

Animals↗

Alternative splicing, expression, and genomic structure of the 3' region of the gene encoding the sarcolemmal-associated proteins (SLAPs) defines a novel class of coiled-coil tail-anchored membrane proteins.

The sarcolemmal associated proteins (SLAPs) are encoded by multiple mRNAs that are presumably generated by alternative splicing mechanisms. The amino acid sequence of the SLAP1 isoform exhibited 76% identity with TOP(AP), a topographically graded antigen of the chick visual system. The regions of coiled-coil structure including an 11-heptad acidic amphipathic alpha-helical segment was conserved with a major divergence in sequence noted in the hydrophobic C termini predicted to be transmembrane domains in the two polypeptides. The genomic organization of the 3' region of the SLAP gene indicated that SLAP1 and TOP(AP) are generated by alternative splicing mechanisms, which are conserved among mammalian and avian species. SLAP1/TOP(AP) were encoded by 11 exons distributed over a minimum of 35 kilobase pairs of continuous DNA; 9 of the exons were constitutively expressed, and 2 were alternatively spliced. The exons range in size from 60 to 321 base pairs, and the predicted functional domains within the polypeptides were encompassed by single exons. The introns vary from 0.2 to 10 kilobase pairs and conform to consensus dinucleotide splicing signals. Reverse transcriptase-polymerase chain reaction studies demonstrated that alternative exons (IV and X) of SLAP were expressed in a tissue-specific fashion and developmentally regulated. The alternatively spliced exon X, which encodes the putative transmembrane anchor in TOP(AP), and a constitutively expressed exon XI, which encodes the putative transmembrane domain in SLAP, were found to target these polypeptides to membrane structures. The presence and conservation of termination codons in exons X and XI render expression of the two SLAP1/TOP(AP) transmembrane domains mutually exclusive. These data reveal that TOP(AP) and SLAP are alternatively spliced products of a single gene that encodes a unique class of tail-anchored membrane proteins.

3' Untranslated Regions↗

Mapping of 34 minisatellite loci resolved by two-dimensional DNA typing.

Two-dimensional (2-D) DNA typing is based on electrophoretic separation of genomic DNA fragments in two dimensions according to independent criteria (size and base-pair sequence), followed by hybridization analysis using multilocus probes. The technique allows simultaneous visualization of several hundred loci as spots in a 2-D pattern. The majority of the loci resolved are polymorphic. Using linkage analysis in a large CEPH family, this study reports the mapping of 34 loci detected by the minisatellite core probe 33.6. By multipoint linkage analysis, regional chromosome positions of the 33.6 loci could be deduced, showing no evidence of clustering. In the analysis of spot patterns, use was made of a computerized image analysis system specifically designed for 2-D DNA typing. Since experimental variations between different separation patterns were automatically corrected for with this program, rapid and reliable scorings could be obtained. The results presented demonstrate the availability of reliable genetic information throughout the 2-D separation pattern. Adding the use of semiautomated computerized pattern analysis, this study further substantiates the applicability of 2-D DNA typing in genome scanning, not only in theoretical but also in practical terms. Moreover, it can be anticipated that this method will have a specific advantage in studies that scan for trinucleotide repeat expansions and somatic instability, where the repeat sequences detected by appropriate core probes are of particular interest.

Chromosome Mapping↗

Demonstration of hepatitis C virus genome in saliva and urine of patients with type C hepatitis: usefulness of the single round polymerase chain reaction method for detection of the HCV genome.

The possible transmission routes of hepatitis C virus (HCV) in patients without overt parenteral exposure (sporadic or community acquired form) were examined. Saliva and urine specimens obtained from type C hepatitis patients, whose sera were positive for the HCV genome, were examined by reverse transcription and polymerase chain reaction (RT-PCR). By analyzing the factors that influenced the detection of the HCV genome by PCR, we developed a single round method which enabled semiquantitative detection with higher sensitivity than that obtained with nested PCR. Single round PCR revealed that 34.8% (8 of 23) of saliva and 56.5% (13 of 23) of urine specimens from patients with type C hepatitis contained the HCV genome. The amounts of HCV genome in saliva and urine specimens correlated with those in serum. The relative amounts of HCV genome in serum, saliva, and urine from a chronic type C hepatitis patient were determined by comparing the reciprocal of the smallest volume of the specimens in which the PCR products were visualized in agarose gels (PCR units/ml), and the values were 1 x 10(5), 5 x 10(1), and 3 x 10(1) PCR units/ml for serum, saliva, and urine specimens, respectively.

Adolescent↗

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi).

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3-crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.

Adaptation, Physiological↗

Multiscale and Bayesian approaches to data analysis in genomics high-throughput screening.

Tremendous amounts of data are produced by high-throughput screening methods currently employed in drug discovery and product development. A typical cDNA microarray or oligonucleotide-based gene chip experiment easily generates over 10,000 data points for each array or chip. The challenge of inferring meaningful information is formidable given the size and number of these datasets. This paper reviews the current status of statistical tools available for gene expression analysis, with emphasis on Bayesian approaches and multiscale wavelet filtering. Fundamental concepts of Bayesian and multiscale modeling are discussed from the perspective of their potential to address important issues related to the analysis of gene expression data, such as the fact that genomic data often have non-Gaussian distributions and feature localization and multiple scales in both frequency and measurement dimension. Recent publications in these areas are reviewed. Wavelet filtering and the advantages of multiscale methods are demonstrated by application to publicly available gene expression data from the National Cancer Institute (NCI). Multiscale methods, including multiscale principal component analysis (MSPCA), are applied to extract gene subsets and to visualize data in multidimensions for comparisons. Similarity in cell lines and gene selection are effectively visualized and quantitatively compared.

Animals↗

ColorHOR--novel graphical algorithm for fast scan of alpha satellite higher-order repeats and HOR annotation for GenBank sequence of human genome.

MOTIVATION: GenBank data are at present lacking alpha satellite higher-order repeat (HOR) annotation. Furthermore, exact HOR consensus lengths have not been reported so far. Given the fast growth of sequence databases in the centromeric region, it is of increasing interest to have efficient tools for computational identification and analysis of HORs from known sequences. RESULTS: We develop a graphical user interface method, ColorHOR, for fast computational identification of HORs in a given genomic sequence, without requiring a priori information on the composition of the genomic sequence. ColorHOR is based on an extension of the key-string algorithm and provides a color representation of the order and orientation of HORs. For the key string, we use a robust 6 bp string from a consensus alpha satellite and its representative nature is tested. ColorHOR algorithm provides a direct visual identification of HORs (direct and/or reverse complement). In more detail, we first illustrate the ColorHOR results for human chromosome 1. Using ColorHOR we determine for the first time the HOR annotation of the GenBank sequence of the whole human genome. In addition to some HORs, corresponding to those determined previously biochemically, we find new HORs in chromosomes 4, 8, 9, 10, 11 and 19. For the first time, we determine exact consensus lengths of HORs in 10 chromosomes. We propose that the HOR assignment obtained by using ColorHOR be included into the GenBank database.

Algorithms↗

SYSTERS, GeneNest, SpliceNest: exploring sequence space from genome to protein.

We have integrated the protein families from SYSTERS and the expressed sequence tag (EST) clusters from our database GeneNest with SpliceNest, a new database mapping EST contigs into genomic DNA. The SYSTERS protein sequence cluster set provides an automatically generated classification of all sequences of the SWISS-PROT, TrEMBL and PIR databases into disjoint protein family and superfamily clusters. GeneNest is a database and software package for producing and visualizing gene indices from ESTs and mRNAs. Currently, the database comprises gene indices of human, mouse, Arabidopsis thaliana and zebrafish. SpliceNest is a web-based graphical tool to explore gene structure, including alternative splicing, based on a mapping of the EST consensus sequences from GeneNest to the complete human genome. The integration of SYSTERS, GeneNest and SpliceNest into one framework now permits an overall exploration of the whole sequence space covering protein, mRNA and EST sequences, as well as genomic DNA. The databases are available for querying and browsing at http://cmb.molgen.mpg.de.

Alternative Splicing↗

Evidence for a DNA replicative genome for RNA-containing tumor viruses.

The requirement for DNA synthesis in the reproduction of RNA tumor viruses was examined using 5-bromodeoxyuridine (BrdU). Addition of BrdU for the first 12 hr after exposure of cells to Rous sarcoma virus resulted in a large decrease in infectious progeny. However, incorporation of [(3)H]uridine into the viral RNA of virions was unaffected. Also, the numbers of virions visualized by electron microscopy were similar in BrdU-treated and untreated samples. Viral group-specific antigen was synthesized after BrdU treatment, and BrdU had little effect on transformation of cells by Rous sarcoma virus. The results support the hypothesis that the replicative genome of RNA-containing tumor viruses contains DNA.

Antigens, Neoplasm↗

The Mycobacterium tuberculosis Transposon Sequencing Database (MtbTnDB): A Large-Scale Guide to Genetic Conditional Essentiality.

Characterizing genetic essentiality across various conditions is fundamental for understanding gene function. Transposon sequencing (TnSeq) is a powerful technique to generate genome-wide essentiality profiles in bacteria and has been extensively applied to Mycobacterium tuberculosis (Mtb). Dozens of TnSeq screens have yielded valuable insights into the biology of Mtb in vitro, inside macrophages, and in model host organisms. Despite their value, these Mtb TnSeq profiles have not been standardized or collated into a single, easily searchable database. This results in significant challenges when attempting to query and compare these resources, limiting our ability to obtain a comprehensive and consistent understanding of genetic conditional essentiality in Mtb. We address this problem by building a central repository of publicly available Mtb TnSeq screens, the Mtb transposon sequencing database (MtbTnDB). The MtbTnDB is a living resource that encompasses to date ≈150 standardized TnSeq screens, enabling open access to data, visualizations, and functional predictions through an interactive web app (www.mtbtndb.app). We conduct several statistical analyses on the complete database, such as demonstrating that (i) genes in the same genomic neighborhood have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles are enriched for genes from similar functional categories. We further analyze the performance of machine learning models trained on TnSeq profiles to predict the functional annotation of orphan genes in Mtb. By facilitating the comparison of TnSeq screens across conditions, the MtbTnDB will accelerate the exploration of conditional genetic essentiality, provide insights into the functional organization of Mtb genes, and help predict gene function in this important human pathogen.

DNA Transposable Elements↗

Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis.

Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed. The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis.

Acute Disease↗