SEARCH · PubMed Health

Results for “sequencing libraries”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,099 records · Page 61Linked to original sources

PubMed1994

Molecular cloning and sequence analysis of the cDNA encoding porcine acrosin inhibitor.

A full-length cDNA encoding the porcine acrosin inhibitor has been isolated from a boar seminal vesicle cDNA library. Nucleotide sequence analysis of the 667-bp cDNA predicts a precursor protein of 97 amino acid residues, which includes a 26-residue signal peptide and a 71-residue secreted protein. The predicted amino acid sequence of the mature protein agrees completely with that of the sperm-associated acrosin inhibitor determined by conventional amino acid sequence analysis. However, the asparagine/aspartic acid and glutamine/glutamic acid substitutions, as reported in the seminal plasma counterpart, have not been observed. Southern blot analysis shows only a single hybridizing band with three different restriction endonucleases, suggesting the presence of a single copy of the acrosin inhibitor gene in the porcine genome.

Acrosin↗

PubMed2004

Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus.

The gene phyA encoding phytase was isolated from Obesumbacterium proteus genomic library and sequenced. The cleavage site of the PhyA signal peptide was predicted and experimentally proved. The PhyA protein shows maximum identity of 53% and 47% to phosphoanhydride phosphorylase from Yersinia pestis and phytase AppA from Escherichia coli, respectively. Based on protein sequence similarity of PhyA and its homologs, the phytases form a novel subclass of the histidine acid phosphatase family. To characterize properties of the PhyA protein, we expressed the phyA gene in E. coli. The specific activity of the purified recombinant PhyA was 310 U mg(-1) of protein. Recombinant PhyA showed activity at pH values from 1.5 through 6.5 with the optimum at 4.9. The temperature optimum was 40-45 degrees C at pH 4.9. The Km value for sodium phytate was 0.34 mM with a Vmax of 435 U mg(-1).

6-Phytase↗

PubMed1995

Construction of YAC contigs at human chromosome 11q22.3-q23.1 region covering the Ataxia telangiectasia locus.

Ataxia telangiectasia (AT) is an autosomal recessive disease of unknown etiology associated with cerebellar ataxia, telangiectasia, immune dysfunction, higher cancer risk, genomic instability and hypersensitivity to ionizing radiation. The major AT loci, AT-A and AT-C, are shown to be closely linked at chromosome 11q22-q23. The most recent genetic linkage mapping and linkage disequilibrium analysis have localized the major AT loci to a sequence of approximately 850 kb between the markers D11S1819 and D11S1818. The isolation of yeast artificial chromosomes spanning the AT region is an essential step to identify the gene or genes responsible for the mutation(s). We isolated a total of 20 YAC clones from three independent YAC libraries, using sequence tagged sites mapped in the AT region as primers for PCR-based YAC screening. The PCR assay for the presence or absence of 16 different DNA markers allowed us to construct and to order four YAC contigs at the AT region. One of the contigs which consists of the 10 YAC clones, covers about 2 Mb of DNA at the boundary between Giemsa-positive band 11q22.3 and Giemsa-negative band 11q23.1 and includes the entire region of the major AT locus between D11S1819 and D11S1818. Thus, the YAC contigs will facilitate the positional cloning approach for searching transcribed sequences from the defined genomic region.

Ataxia Telangiectasia↗

PubMed1995

Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor.

BACKGROUND: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a potent inducer of new blood vessel growth (angiogenesis) that contributes to the pathology of many angiogenesis-associated disease states such as psoriasis, rheumatoid arthritis and cancer. Few molecular entities capable of binding to VPF/VEGF with high affinity and specificity have been described to date. RESULTS: Nuclease-resistant 2'-amino-2'-deoxypyrimidine nucleotide RNA (2'-aminopyrimidine RNA) ligands that bind to VPF/VEGF with high affinity have been identified by iterative rounds of affinity-selection/amplification from two independent random libraries. The sequence information that confers high affinity binding to VPF/VEGF is contained in a contiguous stretch of 24 nucleotides, 5'-CCCUGAUGGUAGACGCCGGGGUG-3' (2'-aminopyrimidine nucleotides are designated with italic letters). Of the 14 ribopurines in this minimal ligand, 10 can be substituted with the corresponding 2'-O-methylpurine nucleotides without a reduction in binding affinity to VPF/VEGF. In fact, the 2'-O-methyl substitution at permissive positions leads to a approximately 17-fold improvement in the binding affinity to VPF/VEGF. The higher affinity results from the reduction in the dissociation rate constant of the 2'-O-methyl-substituted RNA ligand from the protein compared to the unsubstituted ligand. The 2'-O-methyl-substituted minimal ligand, which folds into a bulged hairpin motif, is also more thermally stable than the unsubstituted ligand. Nuclease resistance of the ligand is further improved by the 2'-O-methyl substitutions and the addition of short phosphorothioate caps to the 3'- and 5'-ends. CONCLUSIONS: We have used the SELEX (systematic evolution of ligands by exponential enrichment) process in conjunction with post-SELEX modifications to define a highly nuclease-resistant oligonucleotide that binds to VPF/VEGF with high affinity and specificity.

Animals↗

PubMed1995

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.

Amino Acid Sequence↗

PubMedDate not supplied

Purification, cDNA cloning, and developmental changes in the steady-state mRNA level of rat testicular tissue inhibitor of metalloproteases-2 (TIMP-2).

Using multiple high-performance liquid chromatography (HPLC) steps and high-performance electrophoresis chromatography (HPEC) in conjunction with an [125I]collagen film assay to identify inhibitors of metalloproteases, we have purified a 22-kDa polypeptide to apparent homogeneity from primary Sertoli cell-enriched culture medium. Partial N-terminal amino acid sequence analysis revealed that this protein is similar to the human tissue inhibitor of metalloproteases-2 (TIMP-2). To determine the similarity of rat testicular TIMP-2 to the human homolog, a full-length cDNA coding for rat testicular TIMP-2 was isolated from a rat Sertoli cell cDNA expression library and sequenced. Analysis of the nucleotide sequence and the deduced amino acid sequence of the rat testicular TIMP-2 cDNA revealed an 84 and 98% homology with the human TIMP-2 nucleotide and amino acid sequences, respectively. A survey of its mRNA transcripts in different tissues by northern blots revealed the presence of two mRNA species of 3.7 and 1.3 kb in the testis and brain but not in the kidney, spleen, epididymis, and liver in adult male rats. Studies using polymerase chain reaction (PCR) and Southern blot to detect the TIMP-2 mRNA using total RNA isolated from germ cells, Sertoli cells, and Leydig cells have shown that only Sertoli and Leydig cells expressed TIMP-2 mRNA. These results indicate that Sertoli cells are the major source of TIMP-2 in the testis behind the blood-testis barrier (seminiferous tubule barrier). During testicular development from 3 to 60 days of age, the testicular steady-state TIMP-2 mRNA level increased steadily with an advancement of age. Such an increase in the steady-state testicular TIMP-2 mRNA level apparently is not the result of an up-regulation by germ cells because germ cells cocultured with Sertoli cells failed to elicit an increase in the Sertoli cell steady-state TIMP-2 mRNA level. The results of this study suggest that TIMP-2 secreted by Sertoli cells may play a role in tissue restructuring and germ cell migration during spermatogenesis.

Animals↗

PubMed2006

Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species.

We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S-25S rDNA sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the peri-centromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. GenBank accession numbers: KBrH001P13 (AC 166739); KBrH015B20 (AC 166740); end sequences of KBrH BAC library (CW 978640 - CW 988843); end sequences of KBrS BAC library (DU 826965 - DU 835595); end sequences of KBrB BAC library (DX 010661 - DX 083363).

Brassica↗

PubMed2006

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

Amino Acid Sequence↗

PubMed2001

Heterogeneous nuclear ribonucleoprotein K protein associates with multiple mitochondrial transcripts within the organelle.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein interacts with a subset of cellular RNAs. We used K protein as a bait in the yeast three-hybrid screen to identify RNAs that bind K protein in vivo. A large number of K protein-binding RNA clones were identified from a human hybrid RNA library. These sequences consisted of C-rich patches and were G-poor. Unexpectedly, several of the RNA clones were encoded by the mitochondrial genome. In a subsequent three-hybrid screen of a hybrid RNA library generated from a mouse liver mitochondrial genome, K protein bound RNA sequences encoded by different loci spanning nearly the entire mitochondrial genome. Western blot analysis of extracts from mitochondria and mitochondrial fractions showed that K protein is localized within mitoplasts. Reverse transcriptase PCR of RNA co-immunoprecipitated with K protein from lysates of isolated mitochondria showed that K protein is associated with several processed mitochondrial transcripts. In contrast, in the same assay, the polycistronic nascent mtRNA bound K protein weakly or not at all. Results of this study suggest that K protein acts within functional modules that are responsible for expression of genes in mitochondria.

Animals↗

PubMed1993

Sequence of a cDNA encoding wheat eukaryotic protein synthesis initiation factor 4A.

A cDNA encoding wheat translation initiation factor 4A (eIF-4A) was isolated from a wheat cDNA library and sequenced. The deduced wheat eIF-4A amino acid sequence from the cDNA is compared to eIF-4A from Arabidopsis thaliana, tobacco, mouse and Saccharomyces cerevisiae. Putative RNA helicase motifs, and putative ATP-binding and hydrolysis sites are identified.

Amino Acid Sequence↗

PubMed1993

Two distinct cDNA sequences encoding the human helix-loop-helix protein Id2.

Two distinct Id2 cDNA clones, Id2A and Id2B (inhibitor of differentiation), have been isolated from a human heart cDNA library and sequenced. The deduced amino acid sequences show nearly complete identity, except for the nonsense mutation at codon 37 in Id2B. Characterization of the genomic clones revealed that Id2B is encoded by an intronless gene which probably arose after the evolutionary divergence of rats and humans.

Animals↗

PubMed1991

The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.

Amino Acid Sequence↗

PubMed2000

Cloning, sequence and crystallographic structure of recombinant iron superoxide dismutase from Pseudomonas ovalis.

The gene encoding the iron-dependent superoxide dismutase from Pseudomonas ovalis was cloned from a genomic library and sequenced. The ORF differs from the previously published protein sequence, which was used for the original structure determination, at 16 positions. The differences include three additional inserted residues, one deleted residue and 12 point substitutions. The gene was subcloned and the recombinant protein overexpressed, purified and crystallized in a trigonal space group. The structure was determined by molecular replacement and was refined to 2.1 A resolution.

Amino Acid Sequence↗

PubMed1999

Cloning of novel splice variants of mouse mGluR1.

Three splice variants of the mouse metabotropic glutamate receptor 1, mGluR1E55, mGluR1a and mGluR1b, have been isolated from mouse brain cDNA libraries. The sequences of mGluR1a and mGluR1b are similar to those from rat and human. mGluR1E55 is a novel splice variant. mGluR1E55 has two additional exons. One is 80-bp long at the 5' untranslational region. The other (E55) is 110-bp long at the cysteine-rich region after the ligand-binding domain and before the seven-transmembrane domain. Insertion of the E55 exon results in an inframe stop codon. The predicted protein product contains only the extracellular domain of the receptor and may be secreted.

Alternative Splicing↗

PubMed1995

Identification of a peptide inhibitor against glycosomal phosphoglycerate kinase of Trypanosoma brucei by a synthetic peptide library approach.

A synthetic peptide library, composed of 2.5 million L-amino acid pentapeptides anchored on polystyrene beads was prepared with each bead bearing a single pentapeptide sequence. This library was screened for interaction with glycosomal phosphoglycerate kinase (gPGK) of Trypanosoma brucei labelled with fluorescein or with biotin. Affinity beads that bound the enzyme were selected with a pipette or with streptavidin coated magnetic beads. The beads that bound to the enzyme were individually subjected to Edman microsequence analysis to determine the sequence of the corresponding peptide ligands. The corresponding peptide-sequences were synthesised as free peptide acids and evaluated for enzyme activity inhibition. The pentapeptide NWMMF was able to selectively inhibit gPGK with an IC50 of approximately 80 microM.

Affinity Labels↗

PubMed1991

Cloning and sequencing of the porcine complement factor B.

A genomic clone, SSBf1, containing the complement factor B (BF), a major histocompatibility class III antigen, has been isolated from a porcine genomic library. Partial sequencing and comparison with a human BF gene has identified seven exons coding for amino acids of Ba and Bb, the two subunits of BF. The protein sequence similarity with the human BF is on the average 87%. Southern blot analysis confirmed the existence of only one BF gene per haploid genome. Restriction fragment length polymorphism typing with Taq I showed that there are at least three different porcine BF-haplotypes.

Amino Acid Sequence↗

PubMed1988

Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin).

Amino acid sequences of 285 and 286 residues, respectively, were deduced for mouse and human BM-40 from cDNA clones isolated from expression libraries. The sequences showed 92% identity and were also essentially identical to those of bone osteonectin and of the parietal endoderm protein SPARC. About 60% of the mouse BM-40 sequence was confirmed by Edman degradation. Two of the seven disulfide bonds were localized which apparently separate two distinct domains of mouse BM-40.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of the human melanin-concentrating hormone gene and a variant gene.

Melanin-concentrating hormone (MCH) is a cyclic peptide found expressed almost exclusively in the hypothalamus while MCH-containing fibers project throughout the brain of many vertebrates including man. In fishes, MCH induces melanin concentration within the melanophores and may inhibit ACTH secretion. In mammals, MCH modulates ACTH release in vivo and participates as a neuromediator in the control of complex behaviors such as water and food intake. Salmon, rat and human MCH cDNAs have been cloned and structures of deduced mRNAs and precursors have been elucidated. In this report we determine the nucleotide sequence of two human MCH (hMCH) genes and demonstrate that both genes are expressed in human brain. Cloning from three genomic libraries and sequencing of one class of hMCH genomic DNA reveal high similarity between coding regions and the C-terminal part of the hMCH prohormone. However no sequence identity was found in the N-terminal and 5' end non-coding regions of the gene between them even within 6.5 kilobases (kb) upstream from the truncation point. Using polymerase chain reaction (PCR) analysis we have identified RNA populations that are derived from this gene in human brain. For that reason, this gene is a variant rather than a pseudogene. The authentic hMCH gene could only be cloned by using the PCR technique. With primers specific to 5'-end and 3'-end regions of the MCH mRNA we amplified a 1400 bp fragment as well as other shorter PCR products from human genomic DNA. The longest PCR fragment contains 3 exons encompassing most of the 5' untranslated and all of the coding and 3' untranslated sequences of the hMCH mRNA, that are separated by two introns of 350 and 271 bp, respectively. Interestingly the second intron dissects the hMCH peptide sequence in both the authentic and the variant gene. A strikingly high degree of homology was found between the variant and authentic hMCH genes, including intronic sequences, suggesting that these two genomic sequences diverged very recently during evolution. A strong homology was also noted between the exons and intervening sequences of the human and rat MCH genes. Altogether, our results provide the first strong evidence for the existence of two distinct MCH genes expressing prohormones with different MCH and neuropeptide EI (NEI) sequences in human and along with in vivo and in vitro findings, suggest that these neuropeptides may influence the activity of numerous mammalian neuronal systems.

Amino Acid Sequence↗