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At least 937 records · Page 52Linked to original sources
PubMed2002

Combinatorial approaches: a new tool to search for highly structured beta-hairpin peptides.

Here we present a combinatorial approach to evolve a stable beta-hairpin fold in a linear peptide. Starting with a de novo-designed linear peptide that shows a beta-hairpin structure population of around 30%, we selected four positions to build up a combinatorial library of 20(4) sequences. Deconvolution of the library using circular dichroism reduced such a sequence complexity to 36 defined sequences. Circular dichroism and NMR of these peptides resulted in the identification of two linear 14-aa-long peptides that in plain buffered solutions showed a percentage of beta-hairpin structure higher than 70%. Our results show how combinatorial approaches can be used to obtain highly structured peptide sequences that could be used as templates in which functionality can be introduced.

Amino Acid Sequence↗
PubMed1999

Leishmania: identification of Old World species using a permissively primed intergenic polymorphic-polymerase chain reaction.

We have developed a permissively primed intergenic polymorphic-polymerase chain reaction (PPIP-PCR) which distinguishes between the Old World Leishmania complexes L. major, L. tropica, L. donovani, and L. aethiopica. This technique pairs one parasite-specific and one nonspecific oligonucleotide primer for the PCR. The specific primer was chosen from a unique leishmanial DNA sequence, clone pDOG 2, isolated from a L. donovani chagasi genomic DNA expression library. This sequence has a high DNA homology to the intergenic region of the L. major B/C genes which belong to the polymorphic LmcDNA16 gene family. The specific intergenic primer contains a high GC content, a stem-loop, and a 3'-CG residue. The nonspecific primer was selected from within the pBluescript (SK) plasmid. Using PPIP-PCR, parasites belonging to the L. major, L. tropica, L. donovani, and L. aethiopica complexes could be easily identified directly following agarose gel electrophoresis by the simple profiles of their PCR products. In addition, it was possible to discriminate between strains of L. major or L. donovani from distant geographical regions. Amplification of genomic DNA isolated from several nonleishmanial kinetoplastids yielded either no PCR products or unique bands which were distinct from the leishmanial profiles. Genomic DNA from nonkinetoplastid parasites, plants, or mammals was not amplified by PPIP-PCR. This technique is a rapid and reproducible method for the characterization of Old World Leishmania.

Animals↗
PubMed2004

Construction and analysis of a BAC library in the grass Brachypodium sylvaticum: its use as a tool to bridge the gap between rice and wheat in elucidating gene content.

A BAC library of 30,228 clones with an average insert size of 102 kb was constructed in the grass Brachypodium sylvaticum. Brachypodium has a simple genome, similar in size and repetitive DNA content to that of rice, and is more closely related than rice both to the major temperate cereals wheat and barley, and to the forage grasses. The library represents 6.6 genome equivalents, implying a 99.9% probability of recovering any specific sequence. The library was arrayed onto two high-density colony filters, which were screened with heterologous DNA probes from rice chromosome nine and from syntenous regions of wheat, barley, maize and oat. The construction of Brachypodium BAC contigs revealed that synteny between rice, wheat and Brachypodium was largely maintained over several regions of rice chromosome nine. This suggests that Brachypodium will be a useful tool in the elucidation of gene content in agronomically important cereal crops, complementing rice as a "grass genome model".

Chromosome Mapping↗
PubMed1994

Cloning and sequencing of the cDNA encoding a human testis phospholipid hydroperoxide glutathione peroxidase.

A human cDNA that encodes a polypeptide that has 94% deduced amino-acid sequence identity to porcine phospholipid hydroperoxide glutathione peroxidase was cloned from a testis library. The sequence shows preservation of the UGA selenocysteine codon, putative active-site Trp and Glu residues and a Tyr residue that is phosphorylated in the porcine protein. The 3'-UTR shows some conservation of sequences implicated in the insertion of selenocysteine at an opal codon in human glutathione peroxidase-1.

Amino Acid Sequence↗
PubMed1999

Molecular cloning of a novel alpha2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids.

A novel member of the human CMP-NeuAc:beta-galactoside alpha2, 3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of alpha2, 3-sialyltransferase toward Galbeta1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity, i.e. it utilized Galbeta1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

Amino Acid Sequence↗
PubMed1988

Molecular cloning and chromosomal localization of human membrane cofactor protein (MCP). Evidence for inclusion in the multigene family of complement-regulatory proteins.

Membrane cofactor protein (MCP), a regulatory molecular of the complement system with cofactor activity for the factor I-mediated inactivation of C3b and C4b, is widely distributed, being present on leukocytes, platelets, endothelial cells, epithelial cells, and fibroblasts. MCP was purified from a human T cell line (HSB2) and the NH2-terminal 24-amino acid sequence obtained by Edman degradation. An oligonucleotide probe based on this sequence was used to identify a clone from a human monocytic (U937) cDNA library. Nucleotide sequencing showed a 43-bp 5'-untranslated region, an open reading frame of 1,152 bp, and a 335-bp 3'-untranslated region followed by a 16-bp poly(A) track. The deduced full-length MCP protein consists of a 34-amino acid signal peptide and a 350-amino acid mature protein. The protein has, beginning at the NH2 terminus, four approximately 60-amino acid repeat units that match the consensus sequence found in a multigene family of complement regulatory proteins (C3b-receptor or CR1, C3d-receptor or CR2, decay-accelerating factor, C4-binding protein, and factor H), as well as several other complement and non-complement proteins. The remainder of the MCP protein consists of 25 amino acids that are rich in serine and threonine (probable site of heavy O-linked glycosylation of MCP), 17 amino acids of unknown significance, and a 23-amino acid transmembrane hydrophobic region followed by a 33-amino acid cytoplasmic tail. The MCP gene was localized to human chromosome 1, bands 1q31-41, by analysis of human x rodent somatic cell hybrid clones and by in situ hybridization. This same genetic region contains the multigene family of complement-regulatory proteins, which is thereby enlarged to include the functionally and structurally related MCP.

Amino Acid Sequence↗
PubMed1993

Deletion within the metallothionein locus of cadmium-tolerant Synechococcus PCC 6301 involving a highly iterated palindrome (HIP1).

Genomic rearrangements involving amplification of metallothionein (MT) genes have been reported in metal-tolerant eukaryotes. Similarly, we have recently observed amplification and rearrangement of a prokaryotic MT locus, smt, in cells of Synechococcus PCC 6301 selected for Cd tolerance. Following the characterization of this locus, the altered smt region has now been isolated from a Cd-tolerant cell line, C3.2, and its nucleotide sequence determined. This has identified a deletion within smtB, which encodes a trans-acting repressor of smt transcription. Two identical palindromic octanucleotides (5'-GCGATC-GC-3') traverse both borders of the excised element. This palindromic sequence is highly represented in the smt locus (7 occurrences in 1326 nucleotides) and analysis of the GenBank/EMBL/DDBJ DNA Nucleotide Sequence Data Libraries reveals that this is a highly iterated palindrome (HIP1) in other known sequences from Synechococcus strains (estimated to occur at an average frequency of once every c. 664 bp). HIP1 is also abundant in the genomes of other cyanobacteria. The functional significance of smtB deletion and the possible role of HIP1 in genome plasticity and adaptation in cyanobacteria are discussed.

Amino Acid Sequence↗
PubMed2004

Cloning and characterization of a novel Ca2+/calmodulin-dependent protein kinase I homologue in Xenopus laevis.

In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.

Amino Acid Sequence↗
PubMed1991

Isolation of the spinach nitrite reductase gene promoter which confers nitrate inducibility on GUS gene expression in transgenic tobacco.

Nitrite reductase is the second enzyme in the nitrate assimilatory pathway. The transcription of this gene is regulated by nitrate as well as a variety of other environmental and developmental factors. Genomic clones containing the entire nitrite reductase gene have been isolated from a spinach genomic library and sequenced. The sequence is identical in the transcribed region to a previously isolated spinach NiR cDNA clone (Back et al., 1988) except for the presence of three introns. The analysis of the genomic clones and DNA blot hybridization demonstrates that there is a single NiR gene per haploid genome in spinach. This is in contrast to what has been found for other plant species. The transcription initiation site has been determined by S1 mapping and the 5' upstream region has been used to regulate the GUS reporter gene in transgenic tobacco plants. This gene was found to be regulated by the addition of nitrate in the transgenic plants.

Amino Acid Sequence↗
PubMed2002

Mouse cytosolic acetyl-CoA hydrolase, a novel candidate for a key enzyme involved in fat metabolism: cDNA cloning, sequencing and functional expression.

A cytosolic acetyl-CoA hydrolase (CACH) cDNA has been isolated from mouse liver cDNA library and sequenced. Recombinant expression of the cDNA in insect cells resulted in overproduction of active acetyl-CoA hydrolyzing enzyme protein. The mouse CACH cDNA encoded a 556-amino-acid sequence that was 93.5% identical to rat CACH, suggesting a conserved role for this enzyme in the mammalian liver. Database searching shows no homology to other known proteins, but reveals homological cDNA sequences showing two single-nucleotide polymorphisms (SNPs) in the CACH coding region. The discovery of mouse CACH cDNA is an important step towards genetic studies on the functional analysis of this enzyme by gene-knockout and transgenic approaches.

Acetyl-CoA Hydrolase↗
PubMed2005

Large-scale statistical analysis of secondary xylem ESTs in pine.

A computational analysis of pine transcripts was conducted to contribute to the functional annotation of conifer sequences. A statistical analysis of expressed sequential tags(ESTs) belonging the 7732 contigs in the TIGR Pinus Gene Index (PGI1.0) identified 260 differentially represented gene sequences across six cDNA libraries from loblolly pine secondary xylem. Cluster analysis of this subset of contigs resulted in five groups representing genes preferentially represented in one of the xylem samples (compression wood, plannings, root xylem, latewood) and one group containing mostly genes simultaneously present in compression and side wood libraries. To complement the sequence annotation, 27 cDNA clones representing selected transcripts were completely sequenced. Several genes were identified that could represent putative markers for xylem from different organs, at different stages of development. Several sequences encoding regulatory proteins were over-represented in root xylem as opposed to the other xylem samples. Some of them belonged to known families of plant transcription factors, but two genes were previously uncharacterized in plants. One transcript was homologous to the gene encoding the Smad4 interacting factor, a key co-activator in TGFbeta (transforming growth factor) signalling in animals. Thus, the digital analysis of pine ESTs highlighted a putative gene function of potentially broad interest but that has yet to be investigated in plants. More generally, this study showed that the application of numerical approaches to EST databases should be helpful in establishing priorities among genes to consider for targeted functional studies. Thus, we illustrated the potential of extracting information from conifer sequences already accessible through well-structured public databases.

Amino Acid Sequence↗
PubMed2000

cDNA cloning of cytoplasmic ribosomal protein S7 of winter rye (Secale cereale) and its expression in low-temperature-treated leaves.

A cDNA clone (ScRPS7) encoding the cytoplasmic ribosomal protein S7 was isolated from a rye leaf cDNA library and sequenced. The deduced protein of 192 amino acids with M(r) 22189 shows identity of 52% or 47%, respectively, relative to S7 proteins of human or yeast. A RPS7 mRNA accumulation is higher in the meristematic zone at the leaf base than in the non-meristematic middle and upper section of leaves. Short periods of cold stress sharply reduce the mRNA level while leaves of cold hardened plants contain normal levels of ScRPS7 transcripts.

Amino Acid Sequence↗
PubMed2005

CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome.

An effective tool for the global analysis of both DNA methylation status and protein-chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20,736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein-chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

Base Sequence↗
PubMed1994

Sequence of the 5'-flanking region of the gene encoding human muscle glycogen synthase.

The 5'-flanking region of the gene encoding human muscle glycogen synthase was isolated from a human placental genomic library and sequenced. The sequence is TATA-less and G+C-rich, and putative transcription-controlling sequences were identified. Furthermore, a simple (dC-dA)n sequence repeat was identified about 4 kb upstream from the start codon. This sequence was highly polymorphic and five alleles were typed in the Japanese population using the polymerase chain reaction.

Animals↗
PubMed1990

Nucleotide sequences and novel structural features of human and Chinese hamster hsp60 (chaperonin) gene families.

A number of clones that specifically hybridize to the human hsp60 cDNA (chaperonin protein; GroEL homolog) were isolated from human and Chinese hamster ovary cell genomic libraries. DNA sequence analysis shows that one of these clones, pGem-10, is completely homologous to the human hsp60 cDNA (in both coding and noncoding regions) with no intervening sequences. The other human clones analyzed were all nonfunctional pseudogenes containing numerous small additions, deletions, and base substitutions, but no introns. On the basis of sequence data, six different hsp60 pseudogenes were identified in human cells. In addition, we also cloned and completely sequenced a genomic clone from CHO cells. This clone, which was also a pseudogene, contained a small 87-nucleotide intron near the 3' end. Southern blot analysis of human, mouse, and Chinese hamster DNA, digested with unique restriction enzymes (no sites in cDNA), indicates the presence of about 8-12 genes for hsp60 in the vertebrate genomes. The sequence data, however, suggest that most of these genes, except one (per haploid genome), are likely to be nonfunctional pseudogenes.

Animals↗
PubMed1986

Algorithms for the search of amino acid patterns in nucleic acid sequences.

Some algorithms are described for the search of regions in a nucleic acid sequence that, when translated into amino acids, are homologous to a given amino acid pattern. All algorithms are modifications of the dynamic programming method for sequence comparison such that the translation of codons is taken into account. One of the algorithms has been implemented as a FORTRAN 77 program. The program operates on files that follow the format of the EMBL Nucleotide Sequence Data Library.

Amino Acid Sequence↗
PubMed1994

Molecular structure and organization of the human manganese superoxide dismutase gene.

Human manganese superoxide dismutase (MnSOD) is one of the major cellular defense enzymes that protects against toxic effects of superoxide radicals. Overexpression of human MnSOD has been shown to inhibit radiation-induced neoplastic transformation, suppress malignancy of cancer cells, and increase tolerance to various toxic agents. To elucidate the human MnSOD gene structure for identification of potential regulatory elements, we isolated five lambda clones from a normal human genomic DNA library and sequenced the largest clone containing the entire human MnSOD gene. The results demonstrated that human MnSOD is a single-copy gene consisting of five exons interrupted by four introns with typical splice junctions. A distinctive transcription initiation site was identified 74 bp upstream from the translation start site. This transcription initiation site is preceded by a G + C-rich (78%) promoter region containing a cluster of seven SP1 and three AP2 consensus sequences with no TATA box or CAAT box. The 3'-flanking region of the MnSOD gene contains one NF-kappa B consensus sequence. The presence of SP1, AP2, and NF-kappa B consensus sequences suggests that these potential regulatory elements may play a role in the regulation of human MnSOD gene expression.

Animals↗