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PubMed1994

Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE).

Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.

Base Sequence↗

PubMed1990

Characterization of human cDNA and genomic clones for glial fibrillary acidic protein.

Both a partial cDNA clone and a complete genomic clone have been isolated for human gfa, the gene encoding the major component of astrocyte intermediate filaments, glial fibrillary acidic protein (GFAP). The nucleotide sequence of the entire coding region and 102 bp of the 5' flanking DNA was determined. The mRNA start site was identified by primer extension and probe protection experiments, and a novel in vitro transcription and translation procedure was then used to establish that the first ATG in the mRNA initiates GFAP synthesis. The predicted amino-terminal sequence for human GFAP differs greatly from that previously deduced for mouse GFAP from its gene sequence, despite otherwise high homology. This discrepancy was resolved by determining that the published mouse genomic sequence has an incorrect additional base. The corrected sequence produces strong homology between human and mouse GFAP in their predicted amino acid sequences, and suggests that human and mouse GFAP initiate at homologous positions. The beginning sequence deduced here for both proteins is matched closely by that previously obtained for porcine GFAP by direct sequencing of its amino-terminal end. This supports the protein initiation sites proposed, and also indicates that GFAP is not processed at its amino-terminal end.

Base Sequence↗

PubMed2005

The complete sequence of the bovine torovirus genome.

Viruses in the family Coronaviridae have elicited new interest, with the outbreaks caused by SARS-HCoV in 2003 and the recent discovery of a new human coronavirus, HCoV-NL63. The genus Torovirus, within the family Coronaviridae, is less well characterized, in part because toroviruses cannot yet be grown in cell culture (except for the Berne virus). In this study, we determined the sequence of the complete genome of Breda-1 (BoTV-1), a bovine torovirus. This is the first complete torovirus genome sequence to be reported. BoTV-1 RNA was amplified using long RT-PCR and the amplicons sequenced. The genome has a length of 28.475 kb and consisted mainly of the replicase gene ( approximately 20.2 kb) which contains two large overlapping ORFs, ORF1a and ORF1b, encoding polyproteins pp1a and pp1b, respectively. Sequence analysis identified conserved domains within the predicted sequences of pp1a and pp1b. Sequence alignments and protein secondary structure prediction data suggest the presence of a 3C-like serine protease domain with similarity to the arterivirus 3C-like serine protease and a single papain-like cysteine protease domain with similarity to the picornavirus leader protease. The ADRP (APPR-1'') domain - unique to the Coronaviridae - was also located in BoTV pp1a. In addition, several hydrophobic domains were identified that are typical of a nidovirus replicase. Within the pp1b sequence the polymerase and helicase domains were identified, as well as sequences predicted to be involved in ribosomal frameshifting, including the conserved slippery sequence UUUAAAC and two potential pseudoknot structures.

Amino Acid Sequence↗

PubMed1988

Human ethanol-inducible P450IIE1: complete gene sequence, promoter characterization, chromosome mapping, and cDNA-directed expression.

The human P450IIE1 gene, coding for an ethanol-inducible nitrosamine-metabolizing P-450, was isolated from a lambda EMBL3 genomic library and completely sequenced. The human gene spanned 11,413 base pairs and contained nine exons and a typical TATA box. Upstream and downstream DNAs of 2788 and 559 base pairs were also sequenced and compared to the rat gene. Significant areas of sequence similarity were observed within 140 base pairs upstream of the transcription start site in the rat and human genes. Human DNA 539 base pairs upstream of the transcription start site was inserted into the expression vector pSVOAL delta 5', and luciferase activity was detected when the constructs were introduced into a rat hepatoma cell line. The activity was over 100-fold lower than that of pRSVL, a Rous sarcoma virus LTR-driven luciferase gene. By use of panels of rodent-human cell hybrids, the gene was mapped to chromosome 10 (CYP2E locus). A full-length cDNA, constructed with the first exon of the genomic clone and a partial cDNA clone, was expressed in COS cells and found to code for N-nitrosodimethylamine demethylase activity.

Animals↗

PubMed2025

Characterization of microbial dark matter at scale with MetaSBT and taxonomy-aware Sequence Bloom Trees.

Metagenomics has become a powerful tool for studying microbial communities, allowing researchers to investigate microbial diversity within complex environmental samples. Recent advances in sequencing technology have enabled the recovery of near-complete microbial genomes directly from metagenomic samples, also known as metagenome-assembled genomes (MAGs). However, accurately characterizing these genomes remains a significant challenge due to the presence of sequencing errors, incomplete assembly, and contamination. Here we present MetaSBT, a new tool for organizing, indexing, and characterizing microbial reference genomes and MAGs. It is able to identify clusters of genomes at all seven taxonomic levels, from the kingdom all the way down to the species level, using the Sequence Bloom Tree (SBT) data structure that relies on Bloom Filters (BFs) to index massive amounts of genomes based on their k-mers composition. We have built an initial set of databases composed of over 190 thousand viral genomes from NCBI GenBank and public sources grouped into sequence consistent clusters at different taxonomic levels, making it the first software solution for the classification of viruses at different ranks, including still unknown ones. This results in the definition of over 40 thousand species clusters where ~80% do not match with any known viral species in reference databases to date. Furthermore, we show how our databases can be used as a new basis for existing quantitative metagenomic profilers to unlock the detection of unknown microbes and the estimation of their abundance in metagenomic samples. Finally, the framework is released open-source and, along with its public databases, is fully integrated into the Galaxy Platform enabling broad accessibility.

metagenome-assembled genomes↗

PubMed2002

DNA polymorphisms in ITPA including basis of inosine triphosphatase deficiency.

Intracellular concentrations of the nucleotide inosine triphosphate (ITP) are regulated by ITP-ase (EC 3.6.1.19), which is encoded by ITPA on chromosome 20p. Subjects with complete deficiency of ITP-ase activity (MIM 147520) have elevated ITP concentrations in erythrocytes, but no obvious clinical abnormalities. Based on biochemical screening, complete ITP-ase deficiency has been postulated to result from homozygosity for a dysfunctional allele, with an estimated frequency of 0.05 in Caucasians. ITP-ase deficiency has not yet been characterized at the molecular genetic level. Sequencing of the genomic DNA from a Caucasian subject with complete ITP-ase deficiency revealed homozygosity for missense mutation 198C>A, which predicted a threonine for proline substitution at codon 32 (P32T), whereas among 125 normal Caucasians, there were no homozygotes for P32T (P = 0.0079). The P32T allele frequency of 0.07 in Caucasians was similar to the estimates derived from earlier biochemical studies. P32T was found to be present at varying frequency in other ethnic groups. Two common synonymous single-nucleotide polymorphisms were also identified. These ITPAmarkers, including P32T, provide tools for further study of association with clinical and biochemical phenotypes.

Adult↗

PubMed1993

Characterization of the Schistosoma mansoni gene encoding the glycolytic enzyme, triosephosphate isomerase.

The complete gene encoding Schistosoma mansoni triosephosphate isomerase (TPI) was isolated from a lambda phage genomic library on 2 overlapping clones. These genomic clones have been characterized by restriction mapping and DNA sequencing of the 5' flanking region, the exons, the intron boundaries and the polyadenylation addition site. S. mansoni TPI is encoded by 6 exons spanning a region of about 12 kb. The 5 introns are located at positions precisely analogous to those of mammalian TPI genes but one of the 6 mammalian TPI introns is missing in S. mansoni. We find no evidence of spliced leader involvement in TPI gene expression. The gene is preceded by at least 4 tandem copies of a 2.5-kb repetitive sequence. While the 12-kb size for the S. mansoni TPI gene is much larger than the 3-4 kb typical of mammalian TPI genes, the 42-bp first intron is unusually short. The transcription initiation site for the S. mansoni TPI gene is heterogeneous. Genomic Southern blot analysis suggests that TPI is expressed from a single copy gene.

Amino Acid Sequence↗

PubMed1988

Molecular cloning, complete nucleotide sequence, and gene structure of the provirus genome of a retrovirus produced in a human lymphoblastoid cell line.

We found and characterized a type D retrovirus produced in a human lymphoblastoid cell line of B-cell lineage. The amino acid sequence of the N-terminal region of the purified major structural protein (PVTRSQGQVSSNTTGRASPHPDTHTIPE) revealed no high homology with any of the known retroviral amino acid sequences. We have cloned cDNA and the proviral genome integrated in the retrovirus-producing cells, and determined the complete nucleotide sequence and gene structures of the genome. The provirus genome is 8785 bp long and has the structure of LTR-gag-prt-pol-eny-LTR. The nucleotide sequences of the long terminal repeat (LTR) region and a part of the pol gene were closely related to the available sequences of squirrel monkey retrovirus (SMRV), and we designated this virus SMRVHLB' abbreviated as SMRV-H. The primer (tRNA(Lys)1,2)-binding sequence of SMRV-H (TGGCGCCCAGGACGTGGGGCTCGA) has a GG insertion, which is different from that of SMRV. The transmembrane protein of the 3' terminal region of env gene contains an amino acid sequence of an immunosuppressive peptide (EVVLQNRRGLDLLTAEQGGICLALQERCCFYANKS), in which R is unique in SMRV-H. The core sequence of the glucocorticoid regulatory element is found upstream of the two 42-bp imperfect repeats in the LTR. Sequences partially homologous to those of the rat IgE-binding protein gene are in gag and pol genes.

Amino Acid Sequence↗

PubMed2005

Complete nucleotide sequence and molecular probing of potato virus S genome.

Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).

Carlavirus↗

PubMed2001

A PCR-based assay to detect En/Spm-like transposon sequences in plants.

Degenerate primers deduced from the TPase region of plant En/Spm-like transposons allowed the amplification of similar sequences from various plant species including sugar beet, wheat and pea. These primers are efficient tools for the detection of this family of transposons in many plant genomes irrespective of sequence knowledge or phenotypic pecularities. An efficient PCR assay was therefore developed for these class II transposons, similar to assays already available for Ty1-copia-, Ty3-gypsy- or LINEs. This approach allowed us not only to show the widespread almost-ubiquitous presence of En/Spm-elements in plant genomes, but also to characterize their genomic organization and chromosomal distribution in the genome of chickpea (Cicer arietinum L.) and its abundance in related Cicer species. This approach can be used for the detection and characterization of endogenous DNA transposable elements in plant species, their complete isolation and evaluation of their use for genome analysis.

Amino Acid Sequence↗

PubMed2026

Insights Into the Structural Features, Codon Usage Patterns, and Phylogenetic Analysis in Neoniphon argenteus (Teleostei: Holocentriformes) Based on Complete Mitochondrial Genome.

Neoniphon argenteus, a widely distributed nocturnal coral reef fish in the family Holocentridae, plays an important role in maintaining coral reef ecosystem health, yet its phylogenetic position remains poorly resolved. To bridge this gap, we sequenced and analyzed the complete mitochondrial genome of a specimen from the South China Sea to characterize its structural features, codon usage patterns, and phylogenetic relationships. The 16,569 bp mitogenome (GenBank: PP190474.1) encodes 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs, and two non-coding regions, exhibiting a distinct A + T bias. All tRNAs fold into typical cloverleaf secondary structures except tRNA-Ser (AGN), which lacks the dihydrouridine (DHU) arm. The control region contains palindromic motifs (TACAT/ATGTA) capable of forming hairpin structures and five conserved sequence blocks, whereas the OL region harbors a conserved 5'-GCCGG-3' motif. RSCU analysis revealed 31 frequently used codons (RSCU > 1) with a pronounced preference for A/C-ending codons. The ΔRSCU method identified 10 candidate optimal codons (GCA, CAA, GAA, GGA, AUU, CUA, CCA, CGA, ACA, and GUC). Selection pressure analysis using EasyCodeML and site-specific models indicated that all PCGs are predominantly under purifying selection, with no significant evidence of pervasive positive selection. ND6 exhibited elevated pairwise Ka/Ks ratios (mean = 1.209 ± 0.047), consistent with reduced selective constraint rather than adaptive evolution. Phylogenetic analysis of 19 Holocentriformes species using maximum likelihood and Bayesian inference with partitioned models based on 13 PCGs and two rRNA genes (12S and 16S) assigned all taxa to two well-supported subfamilies (Holocentrinae and Myripristinae). Within Holocentrinae, Neoniphon species form a monophyletic clade nested within a paraphyletic Sargocentron, suggesting that the genus Sargocentron as currently defined is not monophyletic. This study provides useful baseline molecular data for further exploration of the evolutionary history of N. argenteus and other members of Holocentriformes.

Holocentridae↗

PubMed2005

Molecular characterization of a Canadian human parechovirus (HPeV)-3 isolate and its relationship to other HPeVs.

A new member of the Parechovirus genus (HPeV-3) has been recently isolated from a stool specimen of a young Japanese child with transient paralysis (isolate A308/99). Three additional HPeV-3 Canadian cases associated with severe neonatal infections were subsequently described by our group. At this time, limited information is available on the molecular characterization of the HPeV-3 genome. In this study, we report the complete genome sequence of a Canadian HPeV-3 isolate (Can82853-01) recovered in 2001 from the nasopharyngeal aspirate of a neonate with a sepsis-like syndrome. The Can82853-01 genome was 7,322 nucleotides (nt) in length and had 96.4%, 77.7%, and 77.5% nt identity with the A308/99 (HPeV-3), HPeV-1, and HPeV-2 reference strains, respectively. Furthermore, the Can82853-01 polyprotein had 98.2%, 86.9%, and 84.7% amino acids identity compared to the same viruses. Most differences between the polyproteins of HPeV-3 isolates and those of HPeV-1 and -2 were located in the VP0-VP3-VP1 capsid region whereas the non-structural proteins were relatively conserved. The primary and predicted secondary structures of 5'UTR and 3'UTR of the HPeV-3 isolates were also different than those of HPeV-1 and -2 reference strains. Phylogenetic analysis confirmed the relatedness between Can82853-01 and A308/99 and their divergence from other picornaviruses.

3' Untranslated Regions↗

PubMed2002

Gene structure of Taenia solium paramyosin.

Paramyosin is a muscle protein that probably plays a role in the survival of the larval stage of Taenia solium during its prolonged host-parasite relationship. Here we describe the structure of the gene coding for the paramyosin of T. solium. The characterization of two clones obtained from a genomic library showed that the complete gene of paramyosin contains 13 introns delimited by conventional eukaryotic splice signals. Comparison with the paramyosin genes of Drosophila melanogaster and Caenorhabditis elegans showed a lack of conservation of the exon/intron organization in contrast to other muscle genes. No evidence of alternative splicing sites were found, excluding the possibility that T. solium expresses a mini-paramyosin like D. melanogaster.

Amino Acid Sequence↗

PubMed1999

Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita.

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5' region) and a gene for tRNA(Asn) (3' region), and is separated from these genes by two A + T-rich intergenic regions of 1048 (5' region) and 3864 (3' region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3'-->5' exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol gamma in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol gamma from yeast to human.

Agaricales↗

PubMed1990

The mouse keratin 19-encoding gene: sequence, structure and chromosomal assignment.

Keratin 19 (K19) is synthesized mainly in embryonic and adult simple epithelia, but has also been found in stratified epithelia as well. K19 is the smallest known keratin and is remarkable in that, contrary to all other keratins, it does not have a designated partner for the formation of filaments, implying that regulation of its expression is different from other keratin-encoding genes. As a first step in elucidating the mechanisms by which the K19 gene is regulated in relatively undifferentiated embryonic and in terminally differentiated adult tissues, a series of overlapping clones containing the complete mouse K19 gene was isolated from a mouse genomic library and characterized. The nucleotide (nt) sequence extends over 5119 nt and includes six exons. A region of 303 nt upstream from the transcription start point (tsp) was also sequenced. Comparison with the human and bovine K19 genes revealed the existence of homologies in both the coding and noncoding regions. The putative promoter region of the mouse K19 gene is highly homologous to the corresponding sequences of the human and bovine K19 genes. It contains an ATA box, a CAAT box and two potential Sp1-binding sites. Significant homologies were also found between the sequences of the introns of the mouse, human and bovine genes: this was particularly evident in introns 2, 3, 4 and 5. Intron 1, which showed the greatest degree of divergence, was found to contain many repetitive elements. Finally, it is shown that the mouse K19 gene cosegregates with the type-I keratin-encoding gene locus (Krt-1) on chromosome 11.

Amino Acid Sequence↗

PubMed2005

A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma.

Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial rhomboid intramembrane proteases encoded in the recently completed genome of Toxoplasma gondii. Four T. gondii rhomboids (TgROMs) were active proteases with similar substrate specificity. TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite stage responsible for the disease, whereas TgROM2 and TgROM3 were expressed in the oocyst stage involved in transmission. Although both TgROM5 and TgROM4 localized to the cell surface in tachyzoites, TgROM5 was primarily at the posterior of the parasite, whereas adhesins were sequestered in internal micronemes. Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins in a cell-based assay, indicating that it likely provides the key protease activity necessary for invasion. T. gondii rhomboids have clear homologues in other apicomplexans including malaria; thus, our findings provide a model for studying invasion by this deadly pathogen and offer a target for therapeutic intervention.

Animals↗

PubMed2006

DNA motifs in human and mouse proximal promoters predict tissue-specific expression.

Comprehensive identification of cis-regulatory elements is necessary for accurately reconstructing gene regulatory networks. We studied proximal promoters of human and mouse genes with differential expression across 56 terminally differentiated tissues. Using in silico techniques to discover, evaluate, and model interactions among sequence elements, we systematically identified regulatory modules that distinguish elevated from inhibited expression in the corresponding transcripts. We used these putative regulatory modules to construct a single predictive model for each of the 56 tissues. These predictors distinguish tissue-specific elevated from inhibited expression with statistical significance in 80% of the tissues (45 of 56). The predictors also reveal synergy between cis-regulatory modules and explain large-scale tissue-specific differential expression. For testis and liver, the predictors include computationally predicted motifs. For most other tissues, the predictors reveal synergy between experimentally verified motifs and indicate genes that are regulated by similar tissue-specific machinery. The identification in proximal promoters of cis-regulatory modules with tissue-specific activity lays the groundwork for complete characterization and deciphering of cis-regulatory DNA code in mammalian genomes.

Animals↗

PubMed2004

Preference of simple sequence repeats in coding and non-coding regions of Arabidopsis thaliana.

MOTIVATION: Simple sequence repeats or microsatellites have been found abundantly in many genomes. However, the significance of distribution preference has not been completely understood. Completion of the Arabidopsis genome sequencing allows us to better understand and characterize microsatellites. RESULTS: Microsatellite distribution was more abundant in 5'-flanking regions of genes compared with that expected in the whole genome, with an over-representation of AG and AAG repeats; there were clear differences from distributions in 3'-flanks and coding fractions, where triplet frequencies evidently corresponded to codon usage. We identified 1140 full-length genes that contained at least one locus of AG or AAG repeats in their upstream sequences, and whose functional characteristics were significantly associated with the repeats. This observation indicates that selective pressure markedly differed in the three transcribed regions, with positive selection of AG and AAG repeats in 5'-flanks close to those genes whose products are preferentially involved in transcription.

Algorithms↗