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PubMed1999

Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization.

The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization. Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH). In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium. Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E. coli genome, the differences were directly placed in the genome map. About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other. Additional differences in most cases represent sequences that have diverged considerably in the course of evolution. Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species.

Base Sequence↗

PubMed1994

Cloning of a human seven-transmembrane domain receptor, LESTR, that is highly expressed in leukocytes.

Several chemotactic agonists including interleukin-8 (IL-8) and related cytokines have been shown to activate and attract leukocytes via seven-transmembrane domain, GTP-binding protein-coupled receptors. A cDNA clone, LESTR, encoding a protein of 352 amino acids, corresponding to a novel receptor of this type, was isolated from a human blood monocyte cDNA library. The sequence of the deduced protein, LESTR (leukocyte-derived seven-transmembrane domain receptor), has 92.6% identity with that of a recently reported bovine neuropeptide Y (NPY) receptor, boLCR1 (Rimland, J., Xin, W., Sweetnam, P., Saijoh, K., Nestler, E. J., and Duman, R. S. (1991) Mol. Pharmacol. 40, 869-875). LESTR, however, is more similar (> 34%) to the IL-8 receptors, IL-8R1 and IL-8R2, than to several NPY receptors of different origin (< 20%). In the monocyte library, LESTR cDNA fragments were about 20 times as frequent as cDNA coding for IL-8R1 and IL-8R2, and much higher levels of LESTR- than IL-8R-specific mRNA were found in human blood neutrophils and lymphocytes. LESTR transcripts, by contrast, were low or undetectable in several neuroblastoma cell lines that are widely used to study NPY functions. Transfected cells expressing high levels of LESTR mRNA did not bind radiolabeled NPY, IL-8, NAP-2, GRO alpha, PF4, IP10, MCP-1, MCP-3, MIP-1 alpha, HC14, I309, RANTES, C3a, or LTB4. NPY also failed to bind to neutrophils, monocytes, and lymphocytes, to elicit responses in vitro such as Ca2+ changes, shape change, chemotaxis, enzyme release, and the respiratory burst, and to induce leukocyte accumulation upon injection in rats and rabbits. Although the ligand for LESTR could not be identified among a large number of chemotactic cytokines, the high expression in white blood cells and the marked sequence relation to IL-8R1 and IL-8R2 suggest that LESTR may function in the activation of inflammatory cells.

Amino Acid Sequence↗

PubMed2006

Resources for genetic and genomic studies of Xenopus.

The National Institutes of Health Xenopus Initiative is a concerted effort to interact with the Xenopus research community to identify the community's needs; to devise strategies to meet those needs; and to support, oversee, and coordinate the resulting projects. This chapter provides a brief description of several genetic and genomic resources generated by this initiative and explains how to access them. The resources described in this chapter are (1) complementary deoxyribonucleic acid (cDNA) libraries and expressed sequence tag (EST) sequences; (2) UniGene clusters; (3) full-insert cDNA sequences; (4) a genetic map; (5) genomic libraries; (6) a physical map; (7) genome sequence; (8) microarrays; (9) mutagenesis and phenotyping; and (10) bioinformatics. The descriptions presented here were based on data that were available at the time of manuscript submission. Because these are ongoing projects, they are constantly generating new data and analyses. The Web sites cited in each subheading present current data and analyses.

Animals↗

PubMed1992

Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor.

We have isolated the gonadotropin-releasing hormone receptor (GnRH-R) from a rat anterior pituitary cDNA library, determined its sequence and demonstrated receptor function. The 2.2 kb rat GnRH-R clone encodes a protein of 327 amino acids. A 1.3 kb clone encoding the mouse GnRH-R has previously been described (Tsutsumi et al., 1992). Although both the mouse and rat protein share significant homology with molecules belonging to the family of G protein-coupled receptors, they have certain unusual features, an example being the complete absence of a COOH terminal tail. The 3'-untranslated region reported missing in the mouse is present in the rat cDNA, where an extended 1 kb of 3'-untranslated region extending to the poly-A tail is shown. At the amino acid level, the rat GnRH-R shows considerable homology with that of the mouse. Electrophysiological studies with Xenopus oocytes and transfection of the cDNA into COS-1 cells, have shown that the 2.2 kb cDNA clone encodes a functional receptor.

Action Potentials↗

PubMed2005

PAP IB, a new member of the Reg gene family: cloning, expression, structural properties, and evolution by gene duplication.

Reg proteins are expressed in various organs and are involved in cancers and neurodegenerative diseases. They display a typical C-type lectin-like domain but possess additional highly conserved amino acids. By studying human databases and Expressed Sequence Tags library, we identified a new member called PAP IB. Using probabilistic approaches, we established a phylogenetic tree of eighteen Reg proteins. The dendogram showed that they constitute a superfamily composed of three distinct families (FI to FIII) of paralogues that resulted from duplication. We therefore focused on two proteins, REG Ialpha and PAP IB, belonging to the more closely related FI and FII families, respectively. REG Ialpha and PAP IB share 50% sequence identity. After cloning PAP IB, however, we found that it was expressed almost only in pancreas, unlike REG Ialpha, whose expression is ubiquitous. In addition, by building a model of the structure of PAP IB based on the X-ray structure of REG Ialpha, we observed that the two proteins displayed distinctive surface charge distribution, which may lead to different ligands binding. In spite of their common fold that should result in closely related functions, REG Ialpha and PAP IB are a good example of duplication and divergence, probably with the acquisition of new functions, thus participating in the evolution of the protein repertoire.

Amino Acid Sequence↗

PubMed2001

Parasite genome initiatives.

During 1993-1994, scientists from developing and developed countries planned and initiated a number of parasite genome projects and several consortiums for the mapping and sequencing of these medium-sized genomes were established, often based on already ongoing scientific collaborations. Financial and other support came from WHO/TDR, Wellcome Trust and other funding agencies. Thus, the genomes of Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, Leishmania major, Trypanosoma brucei, Brugia malayi and other pathogenic nematodes are now under study. From an initial phase of network formation, mapping efforts and resource building (EST, GSS, phage, cosmid, BAC and YAC library constructions), sequencing was initiated in gene discovery projects but soon also on a small chromosome, and now on a fully fledged genome scale. Proteomics, functional analysis, genetic manipulation and microarray analysis are ongoing to different degrees in the respective genome initiatives, and as the funding for the whole genome sequencing becomes secured, most of the participating laboratories, apart from larger sequencing centres, become oriented to post-genomics. Bioinformatics networks are being expanded, including in developing countries, for data mining, annotation and in-depth analysis.

Animals↗

PubMed2001

Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda.

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).

Amino Acid Sequence↗

PubMed2004

Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose.

In the salvage pathway of GDP-L-fucose, free cytosolic fucose is phosphorylated by L-fucokinase to form L-fucose-L-phosphate, which is then further converted to GDP-L-fucose in the reaction catalyzed by GDP-L-fucose pyrophosphorylase. We report here the cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. Murine L-fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of L-fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP-L-fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP-L-fucose, the reaction product of GDP-L-pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine L-fucokinase and GDP-L-fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of L-fucokinase and GDP-L-fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP-L-fucose in the cytosol.

Alternative Splicing↗

PubMed1999

Analysis of sequence-tagged-connector strategies for DNA sequencing.

The BAC-end sequencing, or sequence-tagged-connector (STC), approach to genome sequencing involves sequencing the ends of BAC inserts to scatter sequence tags (STCs) randomly across the genome. Once any BAC or other large segment of DNA is sequenced to completion by conventional shotgun approaches, these STC tags can be used to identify a minimum tiling path of BAC clones overlapping the nucleation sequence for sequence extension. Here, we explore the properties of STC-sequencing strategies within a mathematical model of a random target with homologous repeats and imperfect sequencing technology to understand the consequences of varying various parameters on the incidence of problem clones and the cost of the sequencing project. Problem clones are defined as clones for which either (A) there is no identifiable overlapping STC to extend the sequence in a particular direction or (B) the identified STC with minimum overlap comes from a nonoverlapping clone, either owing to random false matches or repeat-family homology. Based on the minimum overlap, we estimate the number of clones to be entirely sequenced and, then, using cost estimates, identify the decision rule (the degree of sequence similarity required before a match is declared between an STC and a clone) to minimize overall sequencing cost. A method to optimize the overlap decision rule is highly desirable, because both the total cost and the number of problem clones are shown to be highly sensitive to this choice. For a target of 3 Gb containing approximately 800 Mb of repeats with 85%-90% identity, we expect <10 problem clones with 15 times coverage by 150-kb clones. We derive the optimal redundancy and insert sizes of clone libraries for sequencing genomes of various sizes, from microbial to human. We estimate that establishing the resource of STCs as a means of identifying minimally overlapping clones represents only 1%-3% of the total cost of sequencing the human genome, and, up to a point of diminishing returns, a larger STC resource is associated with a smaller total sequencing cost.

Genome, Human↗

PubMed2002

Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.

Amino Acids↗

PubMed1984

GENAS: a database system for nucleic acid sequence analysis.

A database system, named GENAS (GENe Analyzing System), for computer analysis of sequence was constructed using Adbis which is a relational database management system (1). GENAS enables us to retrieve any sequence data from EMBL nucleotide sequence data library (2) and readily to analyze them (if necessary, together with private data) by various application programs in a interactive manner. Analysis of structure of replication origin of replicons was demonstrated using this system.

Base Sequence↗

PubMed1998

Short-insert libraries as a method of problem solving in genome sequencing.

As the Human Genome Project moves into its sequencing phase, a serious problem has arisen. The same problem has been increasingly vexing in the closing phase of the Caenorhabditis elegans project. The difficulty lies in sequencing efficiently through certain regions in which the templates (DNA substrates for the sequencing process) form complex folded secondary structures that are inaccessible to the enzymes. The solution, however, is simply to break them up. Specifically, the offending fragments are sonicated heavily and recloned, as much smaller fragments, into pUC vector. The sequences obtained from the resulting library can subsequently be assembled, free from the effects of secondary structure, to produce high-quality, complete sequence. Because of the success and simplicity of this procedure, we have begun to use it for the sequencing of all regions in which standard primer walking has been at all difficult.

Animals↗

PubMed1988

Transposed human immunoglobulin V kappa gene regions carry clusters of conserved sequence elements.

The V kappa I gene regions which have been transposed in evolution from the site of the kappa locus on chromosome 2 to chromosomes 1, 22, and other chromosomes, are very similar and may have been derived from one ancestor gene. Upstream from the transposed genes (called orphons) two types of conserved sequence elements were found using a mouse cell assay system. One type is homologous to the murine sequences which were previously thought to be ARS elements; the other one is related to the binding site of the replication/transcription factor NFIII. Such a combination of elements was seen neither in hybridization experiments with the 1 Mb of the kappa locus available on cosmid clones nor in a computer-aided search of sequence data libraries. We speculate that in the evolutionary past, the clustered elements played a role in the transposition of the V kappa genes, perhaps by causing an over-replication and/or by facilitating the integration of the genes.

Base Sequence↗

PubMed1989

Computers: the best friends a human genome ever had.

Mapping and sequencing the human genome will generate large amounts of data, which must be sorted, analyzed, and stored for rapid retrieval to complete this enormous task. Computers and their software programs provide the most important tool to the molecular biologist today. A discussion of current capabilities and future needs in computer hardware and software for the human genome project is the topic of this paper. The use of computer programs to generate restriction maps, manage clone libraries, manage sequence projects, and generate consensus sequences is presented. The use of computers to communicate useful information rapidly to scientific colleagues is also mentioned. The role of both GenBank and BIONET is central to the dissemination and analysis of sequence information. The capabilities of electronic communication worldwide for assisting this project is available on the BIONET National Computer Resource, using existing networks.

Base Sequence↗

PubMed1990

Interspecific comparison of a Drosophila gene encoding FMRFamide-related neuropeptides.

In order to identify functionally important regions of a neuropeptide gene in Drosophila melanogaster, we have studied its occurrence in related species and have characterized the structure of a homologous gene in Drosophila virilis. The melanogaster gene encodes a precursor that contains 13 neuropeptides related to the molluscan tetrapeptide FMRFamide (Nambu et al., 1988; Schneider and Taghert, 1988). Using the melanogaster gene as a probe in Southern blot analysis, related sequences were detected in DNA from each of 7 species tested. D. virilis, which is estimated to have diverged from D. melanogaster between 60 and 80 million years ago (Throckmorton, 1975), was chosen for more detailed study. Immunocytochemical staining using an antibody to authentic FMRFamide revealed a similar set of immunoreactive neurons in the CNS of larvae from the 2 Drosophila species. Using a melanogaster gene probe, overlapping clones were isolated from a virilis genomic library; DNA sequence analysis indicated the presence of a homologous gene. Comparisons of the genes and deduced proteins between the 2 species revealed the following points. (1) Both genes are divided into 2 exons: in D. melanogaster the exons are 106 and 1352 bp long; in D. virilis, they are 169 and at least 1232 bp long; in both species, the intron is approximately 2.5 kb long. (2) The sequence of exon I has largely diverged, and in neither species are exon I sequences translated. In this vicinity of the gene, sequence conservation is limited to a 67 bp region that spans the TATA box and the RNA start site. (3) The deduced neuropeptide precursors have very similar sizes (347 vs 339 amino acids) and the presumed signal sequences are perfectly conserved. (4) While the melanogaster precursor contains 13 FMRFamide-related peptides, the virilis precursor contains only 10. (5) The sequences of some but not all of the FMRFamide-like peptides are perfectly conserved. (6) In the rest of the precursor, significant sequence conservation is found only in the N-terminal portion; immediately downstream of the final FMRFamide-like peptide, the protein sequences are highly divergent. (7) 5' to the RNA start sites (1.2 kb of melanogaster DNA and 1.8 kb of virilis DNA), 17 small (9-52 base pairs) regions are evolutionarily conserved (greater than 80% sequence conservation). We discuss neuropeptide biosynthesis, the functions and evolution of FMRFamide-like neuropeptides in insects, and the cell-specific regulation of neuropeptide gene expression in the contexts of these results.

Animals↗

PubMed2026

Portable metagenomics for preventive surveillance and outbreak control in livestock and poultry: Pathogen detection, resistome profiling, and antimicrobial stewardship.

Conventional diagnostics for livestock and poultry outbreaks commonly rely on culture or targeted PCR panels, which may be too slow or too narrow to guide early control decisions. Portable metagenomics, particularly real-time nanopore sequencing, offers a route to broad pathogen detection, antimicrobial-resistance gene profiling, and outbreak investigation within an integrated workflow. This implementation-focused review evaluates how near-point-of-care metagenomics may support preventive veterinary medicine through earlier detection, surveillance, cohorting, biosecurity decisions, and antimicrobial stewardship. We synthesize sample-to-answer workflows for enteric and respiratory disease in food-producing animals, including sampling, nucleic-acid extraction, host depletion or target enrichment, library preparation, sequencing, bioinformatics, quality control, and interpretation. Applications in calf diarrhea, bovine respiratory disease, poultry outbreaks, mastitis, and resistome monitoring are considered alongside the central limitation that detection alone does not establish causation. Pathogen and resistance-gene signals must therefore be interpreted with clinical signs, lesions, epidemiology, controls, and confirmatory testing. We also propose a minimum reporting checklist, intended as a practical framework rather than a validated consensus standard. Portable metagenomics is not a replacement for conventional diagnostics, but appropriately validated workflows can reduce uncertainty during time-sensitive outbreaks and support more judicious antimicrobial use.

Animals↗

PubMed2003

[Identification of differentially expressed genes in Lin-CD34- and Lin-CD34+ cells].

OBJECTIVE: To identify genes that differentially expressed in Lin(-)CD(34)(-) and Lin(-)CD(34)(+) cells. METHODS: With Lin(-)CD(34)(-) cells as tester and Lin(-)CD(34)(+) cells as driver, cDNA subtractive library for Lin(-)CD(34)(-) cells was constructed using suppression subtractive hybridization technique. Part of clones in the library were sequenced and the homologue analysis was conducted against the DNA database in GenBank. RESULTS: 593 clones containing an average of 300 - 500 bp insert were identified. Of them, 53 randomly selected ESTs were sequenced. Homologue analysis revealed that 37 ESTs represented 10 known genes, and the other 16 ESTs represented 4 novel sequences. CONCLUSION: Part of specifically expressed genes in Lin(-)CD(34)(-) cells were identified, which maybe related to Lin(-)CD(34)(-) cells' specific characteristics.

Antigens, CD34↗

PubMed1993

Molecular analysis of VH and VL regions expressed in IgG-bearing chronic lymphocytic leukemia (CLL): further evidence that CLL is a heterogeneous group of tumors.

We report the heavy (H) and light (L) chain variable (V) region sequences of cDNAs encoding the Ig receptor of two cases of CD5+ IgG-bearing CLL P87 and P103. In both CLL cases the H chain was encoded by members of the VH3 gene family. The L chain expressed by P87 belonged to the V lambda IV subgroup, whereas P103 used a member of the V kappa III subgroup. The VH3.P87 gene differed by only three nucleotides from 38P1, a VH3 gene previously cloned from a fetal liver cDNA library. Nucleotide sequence analysis demonstrated that the V kappa III.P103 gene differed by seven nucleotides from its most homologous germline counterpart, the Humkv325 gene, a highly conserved gene frequently expressed in IgM-bearing CLL. The nucleotide sequences of VH3.P103 and V lambda IV.P87 could not be reliably matched with reported germline V genes. The analysis of multiple independently obtained VH and VL cDNA clones from each tumor showed a lack of intraclonal diversification. The data show that V regions expressed in isotype-switched CD5+ CLL may be either in/near germline configuration or somatically mutated. Furthermore, these tumors, like their IgM-bearing counterparts, do not seem to undergo intraclonal diversification.

Amino Acid Sequence↗