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 1,261 records · Page 70Linked to original sources

PubMed2006

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as -26 degrees C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.

Acclimatization↗

PubMed1995

An arrayed library enriched in hncDNA corresponding to transcribed sequences of human chromosome 19: preparation and analysis.

A simple technique for preparation of libraries of the human chromosome specific transcribed sequences is developed. It uses hnRNA from human-rodent hybrid cell lines containing particular human chromosomes or their fragments and includes three stages: (i) reverse transcription of the hnRNA with Alu-specific primers directing the transcription beyond the Alu-repeats to flanking non-repetitive sequences of the chromosome; (ii) nested primer PCR strategy with specifically designed primers; (iii) direct selective cloning of the second-stage nested primer PCR products. An arrayed hncDNA library was prepared from a hybrid cell line containing chromosome 19 and fragments of 22 and X chromosomes. The library contains around 98% of human-specific transcribed sequences. Sequences of 52 human-specific, according to PCR analysis, clones differed from each other and had no close analogs in the EMBL Data Bank. Of 17 clones assigned to certain human chromosomes, 9 belonged to chromosome 19, 5 to chromosome 22 and 3 to chromosome X. Some of the human specific clones contained repetitive elements scattered over different human chromosomes. Clones from hncDNA libraries are useful as STSs/ESTs, as probes for detecting full-size genes in genomic libraries, for RFLP analysis and for identification of chromosome specific cDNAs.

Animals↗

PubMed1996

Nucleotide sequence of the psbP gene encoding precursor of 23-kDa polypeptide of oxygen-evolving complex in Arabidopsis thaliana and its expression in the wild-type and a constitutively photomorphogenic mutant.

The psbP gene encoding the precursor of 23-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana genomic library and sequenced. The gene harbors three introns and encodes a mature polypeptide of 186 amino acid residues and a transit peptide of 77 amino acid residues. The deduced molecular mass of the mature polypeptide is 23.5-kDa and it contains 22.6% charged amino acid residues which may contribute to the hydrophilic nature of the protein. The transcript encoded by psbP gene of Arabidopsis is approximately 1.3-kb long. In wild-type Arabidopsis seedlings, its expression is organ-specific and is regulated by endogenous developmental cues, light and sucrose. In a constitutively photomorphogenic mutant of Arabidopsis, designated as pho1, the psbP gene is partly derepressed in young, dark-grown seedlings, resulting in a slightly higher level of the transcript. Additionally, the pho1 mutant shows slow accumulation of psbP transcript upon illumination of young, dark-grown seedlings. However, the derepression is not markedly displayed on dark-adaptation of pho1 plants grown in continuous light. These studies, therefore, define the activity of at least one cellular effector involved in regulation of psbP expression.

Amino Acid Sequence↗

PubMed2004

Identification of immune-relevant genes from atlantic salmon using suppression subtractive hybridization.

In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314-BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription-polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.

Aeromonas salmonicida↗

PubMed1996

Construction of a normalized directionally cloned cDNA library from adult heart and analysis of 3040 clones by partial sequencing.

Large-scale sequencing of clones from cDNA libraries derived from specific tissues is a rapid and efficient way of discovering novel genes expressed in those tissues. However, because the heart is continually contracting and relaxing, it strongly expresses muscle-contractile genes and/or mitochondrial genes, a bias that reduces the efficiency of this method. To improve the efficiency of identifying novel genes expressed in the heart, we constructed a normalized directionally cloned cDNA library from adult heart and partially sequenced 3040 clones. Comparisons of these sequence data with known DNA sequences in the database revealed that 57.1% of the clones matched human genes already known, 23.4% were identical or almost identical to human expressed sequence tags (ESTs), 14.2% bore no significant homology to any sequences in the database, and 1.2% represented repetitive sequences. The remaining 4.1% showed some homology with known genes, and Northern blot analysis of several clones in this category revealed that most of them were expressed mainly in the heart and skeletal muscle. After redundancy was excluded, the 3040 clones accounted for 1395 distinctive ESTs, 446 of which exhibited no match to any known sequence. Our results suggest that our normalized library is less redundant than standard libraries and is a useful resource for cataloging genes expressed in the heart.

Adult↗

PubMed2001

Claudin-6: a novel tight junction molecule is developmentally regulated in mouse embryonic epithelium.

Embryonic stem (ES) cells differentiating into embryoid bodies (EBs) have been shown to mimic events of very early development and have become a convenient system in which to identify and study early epithelial specific genes. We describe here the primary structure of a mouse epithelial-specific tight junction gene and its expression patterns in differentiating ES cell-derived EBs in vitro. Sequencing of a clone identified by differential display of 4- vs. 6-day-old EB cells revealed it to overlap exactly with a larger cDNA clone (20M24) that had been isolated, but not characterised, in a screen of an ectodermal library. Complete sequencing and analysis of 20M24 revealed an open reading frame for a 219-amino acid protein with structural features of a transmembrane protein. In cell-free reticulocyte lysates, a 20M24 cDNA corresponding to the open reading frame (660 bp) directed the synthesis of a approximately 23-kDa protein that was localized to cell membranes at cell-cell junctions in transfected HEK-293 cells. Database searches indicated that the cDNA was identical to a recently identified member of the Claudin tight junction family, namely Claudin-6. ES cell cultures were used to further examine the expression pattern of Claudin-6 by whole mount in situ hybridisation during aggregation-induced commitment to epithelial differentiation in vitro. The results indicate that Claudin-6 is one of the earliest molecules to be expressed in ES cells committed to the epithelial fate, and the onset of its expression coincides with the expression of the early epithelial marker, keratin 8 (K8). The initiation of expression of Claudin-6 in vitro is dependent upon plating density as well as serum components. In addition, it was found that Claudin-6 expression is inhibited by Noggin, the Bone Morphogenic Protein (BMP)-signalling pathway inhibitor, suggesting that BMPs may be involved in Claudin-6 expression and epithelialization. These studies establish Claudin-6 as a very early marker of epithelialization and provide evidence that the BMP signalling pathway may be one of the ways that its expression is regulated. These studies also support the power of in vitro ES cell technology to identify and screen novel molecules involved in the early epithelialization of the mouse embryo.

Animals↗

PubMed2005

A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays.

Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here, we introduce a general technique for patterning nanoparticle arrays using two-dimensional crystals of genetically modified hollow protein structures called chaperonins. Constrained chemical synthesis of transition metal nanoparticles is initiated using templates functionalized with polyhistidine sequences. These nanoparticles are ordered into arrays because the template-driven synthesis is constrained by the nanoscale structure of the crystallized protein. We anticipate that this system may be used to pattern different classes of nanoparticles based on the growing library of sequences shown to specifically bind or direct the growth of materials.

Alloys↗

PubMed1986

Mouse thymidylate synthase messenger RNA lacks a 3' untranslated region.

Analysis of the sequence of cDNA corresponding to mouse thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) mRNA revealed that the termination codon TAA was followed immediately by a poly(A) sequence. This raised the possibility that mouse thymidylate synthase mRNA lacks a 3' untranslated region. In the present study, we have further investigated this possibility. DNA corresponding to the 3' end of the thymidylate synthase gene was isolated from a genomic library. The sequence of the genomic DNA was identical to that of the cDNA in the coding region. However, the termination codon was TAG in the genomic sequence rather than TAA, and poly(A) was not present in the genomic DNA. Sequences flanking the site of poly(A) addition were in good agreement with polyadenylylation consensus sequences. S1 nuclease analysis revealed that approximately 80% of the thymidylate synthase mRNA molecules were polyadenylylated at the termination codon. A secondary polyadenylylation site was detected 190-200 nucleotides downstream of the primary site. We conclude that the major species of mouse thymidylate synthase mRNA lacks a 3' untranslated region and that the final A of the termination codon is added by poly(A) polymerase. It appears that a 3' untranslated region is not essential for the accumulation or translation of this mRNA.

Animals↗

PubMed1993

Identification and cloning of GP-3 from rat pancreatic acinar zymogen granules as a glycosylated membrane-associated lipase.

The protein components of highly purified secretory granule membranes and the granule contents from rat exocrine pancreas were characterized by two-dimensional polyacrylamide gel electrophoresis, protein staining, lectin absorption, and Western blotting with anti-secretory protein antibodies. NH2-terminal amino acid sequence was obtained for a approximately 53-kDa glycoprotein denoted GP-3, present only in granule membrane preparations where it was resistant to washing with Na2CO3 and KBr. The sequence of this protein showed homology to pancreatic lipase but was distinct from the NH2-terminal sequence of a 50-kDa content protein presumed to be secretory lipase. Polymerase chain reaction amplification with degenerate oligonucleotide primers to GP-3 and secretory lipase gave partial length subclones that were used to isolate clones from a rat pancreas cDNA library. Dideoxy sequencing of full-length subclones of GP-3 revealed the predicted amino acid sequence for a mature protein of 452 amino acids with a potential N-linked glycosylation site and a deglycosylated molecular weight of 50,860. The GP-3 sequence possesses the serine esterase consensus sequence G-X-S-X-G centered around Ser154 and the catalytic state triad Asp178-His265-Ser154 characteristic of pancreatic lipases. Northern blot analysis of various rat tissues showed GP-3 expression solely in pancreas. Comparison of GP-3 nucleotide and amino acid sequence, along with pancreatic lipases of various species including rat, shows extensive homologies to both proteins and reveals an underlying diversity in the pancreatic lipase family. Close homology is observed between GP-3 and a lipase molecule previously isolated from mouse cytotoxic T cells.

Amino Acid Sequence↗

PubMed2004

Global transcription analysis of immature avian erythrocytic progenitors: from self-renewal to differentiation.

The molecular mechanisms regulating the cell fate decision between self-renewal and differentiation/apoptosis in stem and progenitor cells are poorly understood. Here, we report the first comprehensive identification of genes potentially involved in the switch from self-renewal toward differentiation of primary, non-immortalized erythroid avian progenitor cells (T2EC cells). We used the Serial Analysis of Gene Expression (SAGE) technique in order to identify and quantify the genome fraction functionally active in a self-renewing versus a differentiating cell population. We generated two SAGE libraries and sequenced a total of 37,589 tags, thereby obtaining the first transcriptional profile characterization of a chicken cell. Tag identification was performed using a new relational database (Identitag) developed in the laboratory, which allowed a highly satisfactory level of identification. Among 123 differentially expressed genes, 11 were investigated further and for nine of them the differential expression was subsequently confirmed by real-time PCR. The comparison of tag abundance between the two libraries revealed that only a small fraction of transcripts was differentially expressed. The analysis of their functions argue against a prominent role for a master switch in T2EC cells decision-making, but are in favor of a critical role for coordinated small variations in a relatively small number of genes that can lead to essential cellular identity changes.

Animals↗

PubMed1989

Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae.

Yeast aminopeptidase I is a vacuolar enzyme, which catalyzes the removal of amino acids from the NH2 terminus of peptides and proteins (Frey, J., and Rohm, K-H. (1978) Biochim. Biophys. Acta 527, 31-41). A yeast genomic DNA encoding aminopeptidase I was cloned from a yeast EMBL3A library and sequenced. The DNA sequence encodes a precursor protein containing 514 amino acid residues. The "mature" protein, whose NH2-terminal sequence was confirmed by automated Edman degradation, consists, based only on the DNA sequence, of 469 amino acids. A 45-residue presequence contains positively and negatively charged as well as hydrophobic residues, and its NH2-terminal residues could be arrayed in an amphiphilic alpha-helix. This presequence differs from the signal sequences which direct proteins across bacterial plasma membranes and endoplasmic reticulum or into mitochondria. It remains to be established how this unique presequence targets aminopeptidase I to yeast vacuoles and how this sorting utilizes classical protein secretory pathways. Further, the aminopeptidase I gene, localized previously by genetic mapping to yeast chromosome XI and called the LAP4 gene (Trumbly, R. J., and Bradley, G. (1983) J. Bacteriol. 156, 36-48), was determined by DNA blot analyses to be a single copy gene located on chromosome XI.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation.

Cloning, Molecular↗

PubMed2000

HMMSTR: a hidden Markov model for local sequence-structure correlations in proteins.

We describe a hidden Markov model, HMMSTR, for general protein sequence based on the I-sites library of sequence-structure motifs. Unlike the linear hidden Markov models used to model individual protein families, HMMSTR has a highly branched topology and captures recurrent local features of protein sequences and structures that transcend protein family boundaries. The model extends the I-sites library by describing the adjacencies of different sequence-structure motifs as observed in the protein database and, by representing overlapping motifs in a much more compact form, achieves a great reduction in parameters. The HMM attributes a considerably higher probability to coding sequence than does an equivalent dipeptide model, predicts secondary structure with an accuracy of 74.3 %, backbone torsion angles better than any previously reported method and the structural context of beta strands and turns with an accuracy that should be useful for tertiary structure prediction.

Amino Acid Motifs↗

PubMed2001

Statistical theory for protein combinatorial libraries. Packing interactions, backbone flexibility, and the sequence variability of a main-chain structure.

Combinatorial experiments provide new ways to probe the determinants of protein folding and to identify novel folding amino acid sequences. These types of experiments, however, are complicated both by enormous conformational complexity and by large numbers of possible sequences. Therefore, a quantitative computational theory would be helpful in designing and interpreting these types of experiment. Here, we present and apply a statistically based, computational approach for identifying the properties of sequences compatible with a given main-chain structure. Protein side-chain conformations are included in an atom-based fashion. Calculations are performed for a variety of similar backbone structures to identify sequence properties that are robust with respect to minor changes in main-chain structure. Rather than specific sequences, the method yields the likelihood of each of the amino acids at preselected positions in a given protein structure. The theory may be used to quantify the characteristics of sequence space for a chosen structure without explicitly tabulating sequences. To account for hydrophobic effects, we introduce an environmental energy that it is consistent with other simple hydrophobicity scales and show that it is effective for side-chain modeling. We apply the method to calculate the identity probabilities of selected positions of the immunoglobulin light chain-binding domain of protein L, for which many variant folding sequences are available. The calculations compare favorably with the experimentally observed identity probabilities.

Amino Acid Sequence↗

PubMed1996

Sequence analysis of a cDNA containing the gag and prot regions of the soybean retrovirus-like element, SIRE-1.

SIRE-1 is a family of several hundred dispersed copies of a very large DNA element from Glycine max that has features characteristic of retroviruses and retrotransposons. A 2.4 kb SIRE-1-specific fragment was recovered from a soybean cDNA library and sequenced. The sequence contains two ORFs. Theoretical translation of ORF1 produces a gag-prot-like polyprotein containing highly conserved motifs found in retroelement nucleocapsids (CX2CX4HX4C) and aspartic proteases (LDSG). The second ORF is foreshortened. The cDNA also contains nearly 200 bp of a putative 5' LTR just upstream of a tRNA primer-binding site.

Amino Acid Sequence↗

PubMed1983

Clusters of point mutations are found exclusively around rearranged antibody variable genes.

We have examined the nucleotide sequences of a series of murine antibody genes derived from one kappa light chain gene in order to gain insight into the mechanism that specifically mutates variable genes. Six rearranged VK167 genes from hybridoma and myeloma cells were cloned from bacteriophage lambda libraries. The sequences were compared to the germ-line sequence of the VK167 gene, the JK genes, and the CK gene to identify sites of mutation. Four of six rearranged genes had extensive mutation which occurred exclusively in a 1-kilobase region of DNA centered around the V-J gene. No mutations were found at more distant sites in the intervening sequence or in the constant gene. The frequency of mutation was approximately 0.5% (32 mutations per 6,749 base pairs). Mutations were mostly due to nucleotide substitutions with no preference for transitions or transversions. The location of mutations around each gene indicates that they occur in clusters at random sites. The observation of mutations in the intervening sequence downstream from the JK5 gene rules out models for the mechanism of mutagenesis that rely solely on gene conversion or recombination. The distribution and high frequency of mutations are most easily explained by a mechanism of error-prone repair that occurs during several cycles of cell division.

Animals↗

PubMed1983

Isolation of a genomic clone for bovine pancreatic trypsin inhibitor by using a unique-sequence synthetic DNA probe.

Unique-sequence synthetic DNA probes, based on the known amino acid sequence of bovine pancreatic trypsin inhibitor, were constructed from oligodeoxynucleotides. In genomic Southern blot experiments, these probes were shown to hybridize specifically to discrete restriction fragments. A synthetic probe also was used to isolate a cloned BPTI gene from a bovine genomic library. DNA sequence analysis of this clone indicated that the BPTI coding region was neither preceded by a start codon nor immediately followed by a termination codon. This suggests that the mature form of BPTI may be produced through proteolytic processing from a larger polypeptide precursor.

Amino Acid Sequence↗

PubMed2001

A comprehensive BAC resource.

The Human Genome Project has generated extensive map and sequence data for a large number of Bacterial Artificial Chromosome (BAC) clones. In order to maximize the efficient use of the data and to minimize the redundant work for the research community, The Institute for Genomic Research (TIGR) comprehensive BAC resource (cBACr) (http://www.tigr.org/tdb/BacResource/BAC_resourc e_intro. html) was built as an expansion of the TIGR human BAC ends database. This resource collects, integrates and reports the information on library, maps, sequence, annotation and functions for each human and mouse BAC. The current database contains 635 016 human BACs and 265 617 mouse BACs that were characterized by various approaches, among which 22 705 human clones and 1000 mouse clones have sequence and annotation data.

Animals↗