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PubMed1999

Antigenic properties of peptidic mimics for epitopes of the lipopolysaccharide from Brucella.

The lipopolysaccharide (LPS) is up to now the only identified major virulence determinant of Brucella. This bacterium is responsible for brucellosis in animals and for Malta fever in humans. Several monoclonal antibodies (mAbs) directed against various LPS epitopes have been characterized. Two mAbs, named A15-6B3 and B66-2C8, directed against distinct LPS epitopes have been used to select peptides from 11 phage display libraries. The sequences of the selected peptides contain an overrepresentation of either proline or tryptophan residues when selected with either A15-6B3 or B66-2C8 mAbs, respectively. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed. These data suggest that a subset of the selected peptides are mimotopes of the LPS epitopes.

Amino Acid Sequence↗

PubMed1994

Flavin reductase: sequence of cDNA from bovine liver and tissue distribution.

Flavin reductase catalyzes electron transfer from reduced pyridine nucleotides to methylene blue or riboflavin, and this catalysis is the basis of the therapeutic use of methylene blue or riboflavin in the treatment of methemoglobinemia. A cDNA for a mammalian flavin reductase has been isolated and sequenced. Degenerate oligonucleotides, with sequences based on amino acid sequences of peptides derived from bovine erythrocyte flavin reductase, were used as primers in PCR to selectively amplify a partial cDNA that encodes the bovine reductase. The template used in the PCR was first strand cDNA synthesized from bovine liver total RNA using oligo(dT) primers. A PCR product was used as a specific probe to screen a bovine liver cDNA library. The sequence determined from two overlapping clones contains an open reading frame of 621 nucleotides and encodes 206 amino acids. The amino acid sequence deduced from the bovine liver flavin reductase cDNA matches the amino acid sequences determined for erythrocyte reductase-derived peptides, and the predicted molecular mass of 22,001 Da for the liver reductase agrees well with the molecular mass of 21,994 Da determined for the erythrocyte reductase by electrospray mass spectrometry. The amino acid sequence at the N terminus of the reductase has homology to sequences of pyridine nucleotide-dependent enzymes, and the predicted secondary structure, beta alpha beta, resembles the common nucleotide-binding structural motif. RNA blot analysis indicates a single 1-kilobase reductase transcript in human heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle.

Amino Acid Sequence↗

PubMed1997

Two ldh genes from tomato and their expression in different organs, during fruit ripening and in response to stress.

Two different ldh genes have been isolated from a tomato genomic library and sequenced. Both contain a single intron and correspond to cDNA clones LeLdh1 and LeLdh2 isolated from a library constructed from hypoxically induced tomato roots. Southern blots indicate that the two genes comprise the entire ldh gene family in tomato. Both genes are expressed at low levels in leaves, fruit and roots. Their transcript levels do not change during fruit ripening. Ldh1 but not ldh2 is inducible by oxygen deficit in both roots and fruit.

Base Sequence↗

PubMed1993

Identification of a new family of human epithelial protein kinases containing two leucine/isoleucine-zipper domains.

Using the polymerase chain reaction to study mRNA expressed in human epithelial tumor cells, a member of a new family of protein kinases was identified. The catalytic domain of this kinase has amino-acid-sequence similarity to both the Tyr-specific and the Ser/Thr-specific kinase classes. Clones representing two members of this new family have been isolated from a human colonic epithelial cDNA library and sequenced. The predicted amino-acid sequences of these clones reveal that, in addition to the unusual nature of their kinase catalytic domains, they contain two Leu/Ile-zipper motifs and a basic sequence, near their C-termini. As they possess domains associated with proteins from two distinct functional groups, these kinases have been named mixed-lineage kinases (MLK) 1 and 2. mRNA from MLK1 has been found to be expressed in epithelial tumor cell lines of colonic, breast and esophageal origin. The MLK1 gene has been mapped to human chromosome 14q24.3-31.

Amino Acid Sequence↗

PubMed1995

Isolation and characterization of the gene encoding xylose reductase from Kluyveromyces lactis.

The identification of a xylose reductase (XR)-encoding gene (XYL1) from the xylose-assimilating yeast Kluyveromyces lactis (Kl) is described. XYL1 was isolated as a highly expressed fusion clone from a 'lacZ translational fusion library. DNA sequence analysis revealed an open reading frame (ORF) of 987 bp capable of encoding a polypeptide of 329 amino acids (aa). The deduced aa sequence displays a 62% overall identity to that of XR from Pichia stipitis. Gene disruption studies indicate that XYL1 exists as a single copy in the yeast genome and is essential for growth on xylose. Northern blot analysis of the XYL1 transcript and measurement of the XR enzymatic activities show, in contrast to other known XR-encoding genes, a constitutive expression of Kl XYL1.

Aldehyde Reductase↗

PubMed1992

H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis.

H36 is a 120,000-D membrane glycoprotein that is expressed during the differentiation of skeletal muscle. H36 cDNA clones were isolated from a lambda UniZapXR rat myotube cDNA library and sequenced. The deduced amino acid sequence demonstrates that H36 is a novel integrin alpha chain that shares extensive homology with other alpha integrins that includes: (a) the GFFKR sequence found in all alpha integrins; (b) a single membrane spanning region; (c) conservation of 18 of 22 cysteines; and (d) a protease cleavage site found in the non-I region integrin alpha chains. The cytoplasmic domain of H36 is unique and additional regions of nonhomology further indicate H36 is distinct from all other alpha chains. In keeping with current nomenclature we designate this alpha chain alpha 7. Northern blots demonstrate that expression of H36-alpha 7 mRNA is regulated both early in the development of the myogenic lineage and later, during terminal differentiation. Detection of H36-alpha 7 mRNA coincides with conversion of H36- myogenic precursor cells to H36+ cells. H36-alpha 7 mRNA is present in replicating myoblasts: expression increases upon terminal differentiation and is markedly reduced in developmentally defective myoblasts. In addition, H36-alpha 7 mRNA is not detected in C3H10T1/2 cells. It is in myotubes derived from myoblasts obtained by treatment of 10T1/2 cells with azacytidine or transfection with MRF4. Immunoblots and immunofluorescence demonstrate that the H36-alpha 7 chain is associated with integrin beta 1. Affinity chromatography demonstrates that H36-alpha 7 beta 1 selectively binds to laminin. The expression of H36-alpha 7 on secondary myoblasts during the development of the limb in vivo corresponds with the appearance of laminin in the limb, with the responsiveness of secondary myoblast proliferation to laminin, and with the onset of increased muscle mass, suggesting that H36-alpha 7 modulates this stage in limb development. We conclude that H36-alpha 7 is a novel alpha integrin laminin binding protein whose expression is developmentally regulated during skeletal myogenesis.

Amino Acid Sequence↗

PubMed2004

A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens.

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

Amino Acid Sequence↗

PubMed2006

A permissive secondary structure-guided superposition tool for clustering of protein fragments toward protein structure prediction via fragment assembly.

MOTIVATION: Secondary-Structure Guided Superposition tool (SSGS) is a permissive secondary structure-based algorithm for matching of protein structures and in particular their fragments. The algorithm was developed towards protein structure prediction via fragment assembly. RESULTS: In a fragment-based structural prediction scheme, a protein sequence is cut into building blocks (BBs). The BBs are assembled to predict their relative 3D arrangement. Finally, the assemblies are refined. To implement this prediction scheme, a clustered structural library representing sequence patterns for protein fragments is essential. To create a library, BBs generated by cutting proteins from the PDB are compared and structurally similar BBs are clustered. To allow structural comparison and clustering of the BBs, which are often relatively short with flexible loops, we have devised SSGS. SSGS maintains high similarity between cluster members and is highly efficient. When it comes to comparing BBs for clustering purposes, the algorithm obtains better results than other, non-secondary structure guided protein superimposition algorithms.

Algorithms↗

PubMed2002

Extensive profiling of a complex microbial community by high-throughput sequencing.

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.

Animals↗

PubMed1991

The human homolog of the mouse brown gene maps to the short arm of chromosome 9 and extends the known region of homology with mouse chromosome 4.

The mouse brown locus encodes a tyrosinase-related protein, TRP-1. The human homolog of TRP-1 was recently cloned from a melanoma cDNA library and sequenced. We have made oligonucleotide primers corresponding to the human TRP1 3' untranslated region and used them to map the human TRP1 gene by species-specific PCR in human/rodent somatic cell hybrids. By this means, the human TRP1 gene has been mapped to the short arm of chromosome 9.

Animals↗

PubMed2003

Molecular cloning, sequencing and alternative splicing of the acid phosphatase (Acph) gene from Drosophila virilis.

The Acph gene of Drosophila virilis was isolated from a genomic library and sequenced. The gene was organized into 6 exons interrupted by 5 introns. 5' and 3' RACE identified 3 transcripts ascribed to alternative splicing. One of them coded for 435 amino acid residues; and the others, for 403 amino acids. The shorter ones lacked 34 residues at the C-terminus end encoded by exon 6, which has been implicated in anchoring the protein to the lysosomal membrane.

Acid Phosphatase↗

PubMed1993

Sequence and biological activity of chicken snoN cDNA clones.

cDNA clones of the ski-related gene, sno, were isolated from a chicken cDNA library and sequenced. In contrast to the human system, from which two forms of sno cDNAs have been isolated, we obtained only one type of chicken sno cDNA, that encoding snoN. The coding region for chicken snoN was inserted into the retroviral vectors RCAS(A) and RCASBP(A) and introduced into chicken embryo fibroblasts (CEFs) or quail embryo cells (QECs). Like the various forms of ski, snoN appears to be localized in the nucleus, and high levels of snoN expression cause transformation of CEFs and muscle differentiation of QECs. In contrast to ski however, low-level expression of snoN cannot induce transformation, and is only weakly myogenic.

Amino Acid Sequence↗

PubMed1995

cDNA sequences of variant forms of human placenta diamine oxidase.

Genes for two forms of human placenta diamine oxidase (dao) were cloned from a cDNA library and sequenced. One gene, pdao1, is identical in length to human kidney dao but differs from it by two bases in the coding region and differs slightly in the 3'- and 5'-noncoding regions. The second gene, pdao2, is nearly identical to these genes in the coding region, except that it has an extra 57-nucleotide coding segment near the 3' end of this region. This segment corresponds to the contiguous sequence of the 3' end of intron 3 of human kidney dao.pdao2 also differs significantly from pdao1 and human kidney dao in a 13-base sequence in the 5'-noncoding region. It is proposed that pdao1 and human kidney dao are polymorphic forms of the same allele. Whether pdao2 is a polymorph of these two is not certain, because of the significant differences in the coding and noncoding regions. Pdao2 may represent a different allele.

Amine Oxidase (Copper-Containing)↗

PubMed2005

Expressed sequence tag analysis of zebrafish eye tissues for NEIBank.

PURPOSE: To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by expressed sequence tag (EST) analysis for NEIBank. METHODS: mRNA was extracted from adult zebrafish eye tissues, including lenses, anterior segments (minus lens), retinas, posterior segments lacking retinas, and whole eyes. Five different cDNA libraries were constructed in the pCMVSport6 vector. Approximately 4,000 clones from each library were sequenced and analyzed using various bioinformatics programs. RESULTS: The analysis yielded approximately 2,500 different gene clusters for each library. Combining data from the five libraries produced 10,392 unique gene clusters. GenBank accession numbers were identified for 37.6% (3,906) of the total gene clusters in the combined libraries and approximately 50% were linked to Unigene clusters in the current database. Several new crystallin genes, including two gammaN-crystallins, and a second major intrinsic protein (MIP) were identified in the lens library. In addition, a zebrafish homolog of cochlin (COCH), a gene that may play a role in the pathogenesis of human glaucoma, was identified in the anterior segment library. Surprisingly, no clear ortholog of the major retinal transcription factor Nrl was identified. CONCLUSIONS: The zebrafish eye tissue cDNA libraries are a useful resource for comparative gene expression analysis. These libraries will complement the cDNA libraries made for the Zebrafish Gene Collection (ZGC) and provide an additional source for gene identification and characterization in the vertebrate eye.

Animals↗

PubMed1997

Cloning of GPR37, a gene located on chromosome 7 encoding a putative G-protein-coupled peptide receptor, from a human frontal brain EST library.

A cDNA sequence encoding a putative peptide-specific G-protein-coupled receptor (GPR37) was isolated from a set of human brain frontal lobe expressed sequence tags. The GPR37 cDNA predicts a single open reading frame coding for a 613-amino-acid protein with seven hydrophobic transmembrane domains. The GPR37 genomic sequence was mapped to chromosome 7q31, and it was isolated upon screening of a chromosome 7-specific genomic library. The GPR37 gene spans more than 25 kb and contains two exons and a single intron which interrupts the GPR37 cDNA within the sequence encoding the presumed third transmembrane domain. Northern blot analysis with GPR37 probes revealed a main 3.8-kb mRNA and a less abundant 8-kb mRNA, both expressed in human brain tissues, particularly in corpus callosum, medulla, putamen, and caudate nucleus. The lowest level of expression was detected in cerebellum. The 3.8-kb mRNA is also less abundantly expressed in liver and placenta. Although the ligand for the putative GPR37 receptor has not been identified, its deduced amino acid sequence shows a high degree of homology (approximately 40% in the transmembrane regions) with most mammalian peptide-specific G-protein-coupled receptors and particularly with the human endothelin-B, bombesin-BB1, and bombesin-BB2 receptors.

Adult↗

PubMedDate not supplied

Solution-phase synthesis of a tricyclic pyrrole-2-carboxamide discovery library applying a stetter-Paal-Knorr reaction sequence.

The solution-phase synthesis of a discovery library of 178 tricyclic pyrrole-2-carboxamides was accomplished in nine steps and seven purifications starting with three benzoyl-protected amino acid methyl esters. Further diversity was introduced by two glyoxaldehydes and 41 primary amines. The combination of Pauson-Khand, Stetter, and microwave-assisted Paal-Knorr reactions was applied as a key sequence. The discovery library was designed with the help of QikProp 2.1, and physicochemical data are presented for all pyrroles. Library members were synthesized and purified in parallel and analyzed by LC/MS. Selected compounds were fully characterized.

Amides↗

PubMed1995

A novel stress-inducible metallothionein-like gene from rice.

A novel rice genomic sequence encoding coding segments homologous to other metallothionein-like genes was isolated from Oryza sativa genomic library. This sequence, hereby designated as rgMT (rice genomic metallothionein-like gene), consists of two exons and one intron. From the coding sequence, it is predicted that rgMT encodes one protein of 74 amino acids. Differential expression of rgMT in rice plants was observed as mature transcripts were more abundant in roots than in leaves and sheaths. Under different stress conditions, such as excess heavy metals and heat shock, expression of rgMT was significantly elevated. This was especially noticeable with 250 microM CuCl2 for 16 h, 40 degrees C heat for 2 h and 0.06% DMSO for 1 h. Under sucrose starvation, rgMT transcripts also increased with time up to 72 h. During recovery from sucrose starvation, the transcripts declined slightly within 12 h of recovery. rgMT transcripts were also seen to have increased expression in senescent leaves. These results support the notion that rgMT is a stress-inducible gene in rice heretofore unreported.

Abscisic Acid↗

PubMed2001

The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

Amino Acid Sequence↗