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At least 595 records · Page 33Linked to original sources
PubMed1991

Molecular cloning and nucleotide sequence of the group B streptococcal hemolysin.

Genomic DNA from a Streptococcus agalactiae strain and pUC8 were used to clone the genetic determinant of the group B streptococcal hemolysin. The smallest recombinant plasmid, which enables its E. coli hosts to express beta-hemolysis on sheep blood agar or in blood broth, was found to contain a 3.9 kb insert of streptococcal DNA. A restriction endonuclease map was established for the enzymes Cla I, EcoRI, Hind II, Hind III and Sau I. Transposon mutagenesis experiments with Tn5 extinguished hemolytic activity when Tn5 was inserted at 1.7 kb or 1.9 kb downstream the 5' end of the streptococcal fragment between the Hind II and EcoRI sites. The complete 3.9 kb fragment was sequenced on both strands by the dideoxy method of Sanger. It was shown to contain 4 open reading frames; one of 690 bp, including the area of transposon insertion mentioned above, has a potential promoter region upstream. The deduced amino acid sequence of 230 residues shows a hydrophobic signal sequence of 19 amino acids at its N-terminus.

Amino Acid Sequence↗
PubMed2004

NEMBASE: a resource for parasitic nematode ESTs.

NEMBASE (available at http://www.nematodes.org) is a publicly available online database providing access to the sequence and associated meta-data currently being generated as part of the Edinburgh-Wellcome Trust Sanger Institute parasitic nematode EST project. NEMBASE currently holds approximately 100 000 sequences from 10 different species of nematode. To facilitate ease of use, sequences have been processed to generate a non-redundant set of gene objects ('partial genome') for each species. Users may query the database on the basis of BLAST annotation, sequence similarity or expression profiles. NEMBASE also features an interactive Java-based tool (SimiTri) which allows the simultaneous display and analysis of the relative similarity relationships of groups of sequences to three different databases. NEMBASE is currently being expanded to include sequence data from other nematode species. Other developments include access to accurate peptide predictions, improved functional annotation and incorporation of automated processes allowing rapid analysis of nematode-specific gene families.

Animals↗
PubMed1986

Biosynthetic alr alanine racemase from Salmonella typhimurium: DNA and protein sequence determination.

The nucleotide sequence of the alr gene encoding the biosynthetic alanine racemase in Salmonella typhimurium is reported. The sequence was determined by the dideoxy chain termination method of Sanger mostly from recombinants derived from shotgun and specific subcloning of a 2.6-kilobase region containing the alr gene. The final bridging of nonoverlapping contiguous sequences was accomplished with the use of synthetic site-specific primers. The alr gene was found to be 1077 base pairs in length encoding a protein of 359 amino acid residues. Comparison of alr with the dadB gene encoding the catabolic alanine racemase in S. typhimurium revealed almost identical size (1077 vs. 1068 base pairs) and 52% sequence identity. The respective gene products displayed 43% homology, which includes a decapeptide bearing the pyridoxal 5'-phosphate binding site.

Alanine Racemase↗
PubMed1997

A novel mycobacterial antigen relevant to cellular immunity belongs to a family of secreted lipoproteins.

The gene sequence of a novel 24.1 kDa Mycobacterium tuberculosis protein was identified within the Sanger Centre (UK) M. tuberculosis genome database (cosmid MTCY24G1) by searching with a 126 bp DNA sequence isolated from a genomic M. leprae lambda gt11 library with M. leprae reactive human T cell clones as probes. The 24.1 kDa antigen is common to the vaccine strain Mycobacterium bovis BCG, as well as Mycobacterium leprae. The 699 bp open reading frame encodes a 233 amino acid long precursor protein with a signal peptide sequence for secretion and a consensus motif for lipid conjugation, which suggests that the mature protein is an exported lipoprotein antigen. The molecular mass of the mature protein antigen from M. leprae sonicate was shown to correspond to the deduced size of the M. tuberculosis protein by T cell Western analysis. Homology searches revealed two other similarly sized hypothetical secreted mycobacterial lipoproteins within the M. tuberculosis genome database.

Amino Acid Sequence↗
PubMed1995

Ultrasensitive near-IR fluorescence detection for capillary gel electrophoresis and DNA sequencing applications.

Electropherograms of oligonucleotides labeled with near-IR fluorescent dyes, separated by capillary gel electrophoresis and detected using an ultrasensitive near-IR fluorescence detection system, are presented. A universal M13 sequencing primer was labeled on the 5' end with a near-IR dye containing an isothiocyanate functional group. Comparison of the on-column detection limits in capillary gel electrophoresis for the near-IR dye-labeled sequencing primer to those obtained for a visible fluorescein-labeled primer indicated improved sensitivity for the near-IR case. The detection limit was found to be 3.4 x 10(-20) mol (SNR = 3) for the near-IR dye-labeled primer, while the on-column detection limit for the fluorescein analog was 1.5 x 10(-18) mol (SNR = 3). The sequence of nucleotide bases in an M13mp18 template was determined using a single lane, single dye technique. The molar concentrations of the ddNTPs used during chain extension reactions were varied to achieve a ratio of 4:2:1:0 (A:C:G:T), which allowed the identification of each terminal base via fluorescence intensity measurements. Sequencing ladders were prepared from the M13mp18 template using standard Sanger dideoxy chain-terminating techniques, the modified T7 DNA polymerase, and the near-IR dye-labeled M13 universal primer. The data indicated reliable sequence determination by the 4:2:1:0 (A:C:G:T) peak height identification method up to 250 bases from the annealing site. Comparison of the known sequence of the M13mp18 plasmid to that obtained using this protocol yielded a base-calling accuracy of 84% for the 4:2:1:0 ratio.

Base Sequence↗
PubMed2002

Sequencing the Aspergillus fumigatus genome.

Aspergillus fumigatus is the most common mould pathogen of human beings and unusually causes both invasive disease in immunocompromised patients and allergic disease in patients with atopic immune systems. 4% of patients dying in modern European teaching hospitals have invasive aspergillosis and it is the leading infectious cause of death in leukaemia and bone marrow transplant patients. Until 2001, only two licensed antifungal drugs were available to treat aspergillosis-amphotericin B and itraconazole. Its 28-30Mb genome is being sequenced in an international collaboration, with the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA) as the two main centres. A whole-genome shotgun approach was adopted and initiated in 2001 with an expected completion date in 2003. The complete sequence will permit identification of pathways specific to pathogenic Aspergillus species, help identify new targets for antifungal drugs, and enable investigations into the basic biology of fungi. Numerous secondary metabolic pathways with biotechnological applications and pharmacological properties are found in the Aspergilli and the genome sequence will facilitate research in this area.

Amphotericin B↗
PubMed2023

The genome sequence of a caddisfly, Limnephilus auricula (Curtis, 1834).

We present a genome assembly from an individual female Limnephilus auricula (a caddisfly; Arthropoda; Insecta; Trichoptera; Limnephilidae). The genome sequence is 971.3 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 18.29 kilobases in length.

Limnephilus auricula↗
PubMed1989

New technologies for large-genome sequencing.

Interest in DNA sequencing has increased in recent years and research into new instrument systems to optimize and automate the process has been intensified. A number of commercial devices, from Europe, Japan, and the United States, which instrument aspects of (mainly) the Sanger method, are now available and several new DNA sequencing techniques have been reported. This review briefly describes current methods of DNA sequencing and provides an account of systems presently available or under development for instrumenting the technology. The problems of automation for large-scale DNA sequencing are discussed.

Automation↗
PubMed2002

Genomic evidence for mating abilities in the asexual pathogen Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. It is classified as asexual because no direct observation of mating or meiosis has been reported. Sequencing of the complete genome by an international collaboration, including the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA), has made most of the genomic sequence information from A. fumigatus publicly available. By searching the incomplete genome sequence of A. fumigatus, I have identified the coding capacity for a set of proteins that could be involved in mating and the pheromone response pathway. These include one putative mating-type gene, one gene encoding a pheromone and two pheromone-receptor genes. The mating-type gene encodes a high-mobility group domain protein exhibiting significant similarity with mating-type proteins from sexually reproducing filamentous ascomycetes. The pheromone gene is predicted to encode a precursor pheromone that is processed by a KEX2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. In addition, the deduced gene products of the receptor genes are putative seven-transmembrane proteins, which display a high-level amino acid identity with the a-factor receptor Ste3p and the alpha-receptor Ste2p of S. cerevisiae, respectively. The identification of these homologues suggests the existence of a sexual cycle in A. fumigatus.

Amino Acid Sequence↗
PubMed1982

A new aspect of a restriction endonuclease Tth111 I. It has a degenerated specificity (Tth111 I).

We previously reported that Thermus thermophilus 111 contained two restriction enzymes, Tth111 I and Tth111 II. The former does not cleave phi X174RFDNA and the latter does. We have now found another endonuclease activity able to cleave phi X174RFDNA in the cell extract of T. thermophilus 111. The protein with this activity was purified in a homogeneous state by chromatography on cellulose phosphate, heparin-Sepharose 4B and hydroxylapatite, successively. However, this endonuclease activity was always accompanied with Tth111 I activity during the purification procedure and the purified protein also showed a strong Tth111 I activity, suggesting that the Tth111 I activity and the phi X174RFDNA-cleaving activity reside in a single molecule. The phi X174RFDNA-cleaving activity was enhanced more strongly with Mn2+ than with Mg2+ and seemed to be attributable to a relaxed specificity of Tth111 I activity as seen in the cases of EcoRI* and BamHI* Thus we designated the phi X174RFDNA-cleaving activity Tth111 I*. The molecular weight of the protein with both Tth111 I and Tth111 I* activities was determined to be about 76,000 by gel filtration on a Sephadex G-100 column and 39,000 by SDS-polyacrylamide gel electrophoresis, suggesting the enzyme to be a dimer consisting of identical polypeptide chains. The phi X174RFDNA sequences surrounding Tth111 I* cuts were determined by the chain terminator method of Sanger et al. The results confirmed that Tth111 I* recognized a degenerated form of the Tth111 I recognition sequence, i.e., a sequence such that one of the specified nucleotides in the Tth111 I recognition sequence, 5'GACNNNGTC3', was substituted with N (N stands for any of A, G, C, and T), such as 5'NACNNNGTC3', 5'GACNNNNTC3', 5'GACNNNGNC, and so on (arrows indicate cleavage sites).

Bacterial Proteins↗
PubMed2026

ERGA-BGE reference genome of the Eurasian Woodcock ( Scolopax rusticola), a game bird species with isolated populations of conservation interest.

The reference genome of the Eurasian Woodcock ( Scolopax rusticola) is an important resource to investigate population structure across the wide breeding range of this iconic game species and the conservation status of specific management units, such as the isolated Macaronesian populations. The genome sequence was assembled into 45 contiguous chromosomal pseudomolecules and 2 sex chromosomes (W and Z). This chromosome-level assembly encompasses 1.2 Gb, composed of 1,613 contigs and 935 scaffolds, with contig and scaffold N50 values of 5.9 Mb and 34.2 Mb, respectively.

Aves↗
PubMed1993

No evidence for oncogenic mutations in guanine nucleotide-binding proteins of human adrenocortical neoplasms.

G-Proteins are membrane-bound heterotrimeric polypeptides that couple receptor signals to second messenger systems such as cAMP. Recently, point mutations at 2 codons of the highly preserved alpha-chain of Gs, the adenyl cyclase-stimulating G-protein, were found in GH-secreting pituitary tumors. These mutations resulted in constitutively activated Gs alpha and high intracellular cAMP levels. In addition, point mutations at similar codons of a different G-protein, G(i) alpha 2, were reported in adrenocortical neoplasms, suggesting a potential role of this isoform in the genesis of these tumors. We reevaluated the frequency of constitutively activating point mutations in the alpha-chain of the stimulatory (Gs alpha) and inhibitory (G(i) alpha 2) G-proteins in human adrenocortical tumors. Seven adrenocortical carcinomas, 2 human adrenocortical tumor cell lines, and 11 adrenocortical adenomas were studied. Genomic DNA was purified from either frozen tumor tissue or paraffin-embedded sections. Using specific primers and the polymerase chain reaction, DNA fragments surrounding codons 201 and 227 (Gs alpha) and 179 and 205 (G(i) alpha 2) were amplified and visualized on a 2% agarose gel. In a second asymmetric polymerase chain reaction, using nested primers, single stranded DNA was generated using 1-10 microL of the initial amplification mixture and directly sequenced using the dideoxy chain termination method of Sanger. We found no mutations at codons 201, 227 and 179, 205 of Gs alpha and G(i) alpha 2, respectively, in the tumors studied. We conclude that previously identified oncogenic point mutations in the stimulatory and inhibitory alpha-chain of G-proteins do not appear to be present at high frequency in adrenal neoplasms. Thus, the mechanism(s) of tumorigenesis in these tumors is different from that in GH-secreting adenomas and may involve oncogenic mutations of other cell constituents.

Adolescent↗
PubMed1995

Gene transcripts of eleven proteins with specific functions are all detected in human normal cells and tumor cell lines: a possible DNA-->RNA basal constant flow.

Existance of messenger ribonucleic acids of eleven functionally differentiated proteins in normal human cells and tumor cell lines was investigated using reverse transcription--nested polymerase chain reaction method. Examined gene transcripts were those of progesterone receptor, estrogen receptor, interleukin 2, CD8, parathyroid hormone, cholecystokinin/pancreozymin, glucagon, insulin, adrenocorticotropic hormone, enkephalin and thyroid stimulating hormone. In RT-PCR almost all primers were originally designed and sequences of PCR products were confirmed by the Sanger's method. Investigated cells were normal human peripheral mononuclear cells and their subsets, lymphokine-activated killer cells, gastric mucosal cells, sperm and fifteen established tumor cell lines. All of the examined mRNAs were detected in all of the above cell groups. Therefore, cells of independent tissues or tumors share same kinds of mRNA such as steroid hormone receptors, cytokine, lymphocyte surface molecule and parathyroid, digestive and cerebral hormones. These findings strongly suggest that every cell can express every mRNA. Beneath the cell differentiation there may exist a DNA-->RNA basal constant flow, the arrow of time captured in a cell.

Base Sequence↗
PubMed1996

Partial characterization of zeugmatin indicates that it is part of the Z-band region of titin.

Zeugmatin is a muscle specific protein discovered by Maher et al. [1985: J. Cell Biol. 101:1871-1883] to be in Z-Bands of muscle and in the dense bodies of smooth muscle. Maher et al. [1985] generated a zeugmatin specific monoclonal antibody, McAb20, and then used immunoaffinity chromatography to isolate a 600-800 kD protein. During myofibrillogenesis of embryonic cardiac muscle, zeugmatin is detected in fully formed Z-bands in the mature myofibrils but not in the Z-bodies of premyofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24]. Rhee et al. [1994] have postulated that zeugmatin may be responsible for the fusion of the alpha-actinin containing Z-bodies to form the solid Z-Bands of the mature myofibrils. The current studies were undertaken to characterize the properties of zeugmatin. The McAb20 was used to probe a chicken heart lamba gt11 expression library, and three unique positive clones of 1.1, 1.4, and 1.7 kB were isolated. These were inserted into pcDNA3, sequenced, and assembled into a 1.8 kB ORF. A 60% identity with N-terminal region of the human cardiac titin sequence was revealed at the amino acid level. This region of the 1.8 kB zeugmatin sequence is located entirely in the Z-band region of the human cardiac titin molecule. The 1.1 kB clone of zeugmatin was subcloned into pTrcHisC and expressed in bacteria. Bacterial lysates were prepared and run over nickel columns to isolate a 46 kD fusion protein. This fusion protein formed a complex with purified alpha-actinin that could be immunoprecipitated with the zeugmatin specific antibody, McAb 20. The 1.1 kB sequence was transfected into non-muscle cell lines, PtK2 and REF. Twenty-four hours after transfection, the 46 kD zeugmatin peptide, not present in control non-muscle cells, was localized in focal adhesions and in a punctate pattern along the stress fibers. Double immunofluorescence staining revealed that zeugmatin colocalized with the alpha-actinin in the dense bodies and focal contacts of the stress fibers. At longer time points, as the transfected cells accumulated more truncated zeugmatin molecules, the cells lost adhesion plaques and stress fibers, and became detached from the substratum. Our results indicate the zeugmatin is part of the titin molecule that is located within the Z-band and that this section of the titin molecule anchors the actin crosslinking alpha-actinin molecules.

Actinin↗
PubMed1989

1-Tuple DNA sequencing: computer analysis.

A new method of DNA reading was proposed at the end of 1988 by Lysov et al. According to the authors' claims it has certain advantages as compared to the Maxam-Gilbert and Sanger methods, which are revealed by automation and rapidity of DNA sequencing. Nevertheless its employment is hampered by a number of biological and mathematical problems. The present study proposes an algorithm that allows to overcome the computational difficulties occurring in the course of the method during reconstruction of the DNA sequence by its l-tuple composition. It is shown also that the biochemical problems connected with the loss of information about the l-tuple DNA composition during hybridization are not crucial and can be overcome by finding the maximal flow of minimal cost in the special graph.

Algorithms↗
PubMed2001

Molecular cloning of novel mouse and human putative citrate lyase beta-subunit.

Using a fluorescent differential display (FDD) technique, a novel cDNA was identified by screening for gene expressed differentially between the Dunn osteosarcoma cell line and the LM8 cell line, an isolated variant of the Dunn cell line that has high metastatic potential to the lung. Molecular cloning of the cDNA revealed the clone has similarity to a bacterial fermentation enzyme, the citrate lyase beta-subunit (CL-beta). Northern blot and competitive reverse transcription-PCR (RT-PCR) analysis revealed up-regulation of the gene in the LM8 cell line. An RNA Master blot indicated that the mRNA encoding CL-beta is expressed abundantly in murine heart, liver, and kidney. A human expressed sequence tag (EST) database search suggested that a similar cDNA is expressed in humans. A gene with identical sequence is located on chromosome 13 in the genome database (Sanger centre, UK). These data suggest that a citrate fermentation pathway may exist in eukaryotes including mammals.

Animals↗