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PubMed1995

MHV-3 induced prothrombinase is encoded by musfiblp.

Previously, we demonstrated induction of a unique macrophage prothrombinase, PCA, in MHV-3 infected BALB/cJ mice. By immunologic screening, a clone representing PCA was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp, but when infected with MHV-3, synthesized musfiblp-specific mRNA. Musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrate when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.

Animals↗
PubMed1999

Expression of a novel member of the TGF-beta superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues.

We have cloned a novel member of the transforming growth factor-beta (TGF-beta) superfamily from a human placental cDNA library. The sequence is identical to five very recently published sequences, of which only one (macrophage inhibitory cytokine-1, MIC-1) has been characterized in terms of function. In light of the present data demonstrating the wide distribution of the mRNA and putative multifunctionality, we propose to name this molecule growth/differentiation factor-15/MIC-1 (GDF-15/MIC-1). The deduced amino acid sequence reveals typical features of a secreted molecule. The epithelium of the choroid plexus is the only site in the adult brain expressing detectable levels of GDF-15/MIC-1 mRNA. Many epithelia of non-neural tissues including those of the prostate and intestinal mucosa, bronchi and bronchioli, secretory tubuli of the submandibular gland, and lactating mammary gland are prominent sites of GDF-15/MIC-1 synthesis. GDF-15/MIC-1 is also strongly expressed by macrophages in the adrenal gland. Thus, GDF-15/MIC-1, like many other members of the TGF-beta superfamily, is widely distributed in adult tissues, being most strongly expressed in epithelial cells and macrophages.

Animals↗
PubMed2006

Nuclear receptors of the honey bee: annotation and expression in the adult brain.

The Drosophila genome encodes 18 canonical nuclear receptors. All of the Drosophila nuclear receptors are here shown to be present in the genome of the honey bee (Apis mellifera). Given that the time since divergence of the Drosophila and Apis lineages is measured in hundreds of millions of years, the identification of matched orthologous nuclear receptors in the two genomes reveals the fundamental set of nuclear receptors required to 'make' an endopterygote insect. The single novelty is the presence in the A. mellifera genome of a third insect gene similar to vertebrate photoreceptor-specific nuclear receptor (PNR). Phylogenetic analysis indicates that this novel gene, which we have named AmPNR-like, is a new member of the NR2 subfamily not found in the Drosophila or human genomes. This gene is expressed in the developing compound eye of the honey bee. Like their vertebrate counterparts, arthropod nuclear receptors play key roles in embryonic and postembryonic development. Studies in Drosophila have focused primarily on the role of these transcription factors in embryogenesis and metamorphosis. Examination of an expressed sequence tag library developed from the adult bee brain and analysis of transcript expression in brain using in situ hybridization and quantitative RT-PCR revealed that several members of the nuclear receptor family (AmSVP, AmUSP, AmERR, AmHr46, AmFtz-F1, and AmHnf-4) are expressed in the brain of the adult bee. Further analysis of the expression of AmUSP and AmSVP in the mushroom bodies, the major insect brain centre for learning and memory, revealed changes in transcript abundance and, in the case of AmUSP, changes in transcript localization, during the development of foraging behaviour in the adult. Study of the honey bee therefore provides a model for understanding nuclear receptor function in the adult brain.

Amino Acid Sequence↗
PubMed2005

High-throughput discovery of germ-cell-specific genes.

As a specialized cell population that plays the unique role of transmitting genetic information to subsequent generations, germ cells have been intensively studied to unravel their unique physiology from the specification of primordial germ cells to the fateful reunion of gametes during fertilization. For their differential expression, germ-cell-specific genes are the keys to understanding these unique features. In the last decade, the emerging methodologies designed for large-scale and high-throughput analysis have created an ever-increasing amount of data. Among these methodologies, expressed sequence tag libraries, serial analysis of gene expression, and microarrays provide valuable expression data that can be further analyzed. Using the mouse as a model system, we describe a strategy starting from the quick identification of germ-cell-specific genes using public domain expression data to the functional characterization of the identified genes using targeted gene disruption. This strategy should accelerate the process to fill in the missing pieces of the germ cell physiology puzzle and the construction of genetic networks to help us to understand the etiology of infertility. Furthermore, these identified germ-cell-specific genes may lead to the development of new contraceptives targeted specifically to germ cells.

Animals↗
PubMed2007

Recent proliferation and translocation of pollen group 1 allergen genes in the maize genome.

The dominant allergenic components of grass pollen are known by immunologists as group 1 allergens. These constitute a set of closely related proteins from the beta-expansin family and have been shown to have cell wall-loosening activity. Group 1 allergens may facilitate the penetration of pollen tubes through the grass stigma and style. In maize (Zea mays), group 1 allergens are divided into two classes, A and B. We have identified 15 genes encoding group 1 allergens in maize, 11 genes in class A and four genes in class B, as well as seven pseudogenes. The genes in class A can be divided by sequence relatedness into two complexes, whereas the genes in class B constitute a single complex. Most of the genes identified are represented in pollen-specific expressed sequence tag libraries and are under purifying selection, despite the presence of multiple copies that are nearly identical. Group 1 allergen genes are clustered in at least six different genomic locations. The single class B location and one of the class A locations show synteny with the rice (Oryza sativa) regions where orthologous genes are found. Both classes are expressed at high levels in mature pollen but at low levels in immature flowers. The set of genes encoding maize group 1 allergens is more complex than originally anticipated. If this situation is common in grasses, it may account for the large number of protein variants, or group 1 isoallergens, identified previously in turf grass pollen by immunologists.

Antigens, Plant↗
PubMed2006

Thauera and Azoarcus as functionally important genera in a denitrifying quinoline-removal bioreactor as revealed by microbial community structure comparison.

Structural shifts associated with functional dynamics in a bacterial community may provide clues for identifying the most valuable members in an ecosystem. A laboratory-scale denitrifying reactor was adapted from use of non-efficient seeding sludge and was utilized to degrade quinoline and remove the chemical oxygen demand. Stable removal efficiencies were achieved after an adaptation period of six weeks. Both denaturing gradient gel electrophoresis profiling of the 16S rRNA gene V3 region and comparison of the 16S rRNA gene sequence clone libraries (LIBSHUFF analysis) demonstrated that microbial communities in the denitrifying reactor and seeding sludge were significantly distinct. The percentage of the clones affiliated with the genera Thauera and Azoarcus was 74% in the denitrifying reactor and 4% in the seeding sludge. Real-time quantitative PCR also indicated that species of the genera Thauera and Azoarcus increased in abundance by about one order of magnitude during the period of adaptation. The greater abundance of Thauera and Azoarcus in association with higher efficiency after adaptation suggested that these phylotypes might play an important role for quinoline and chemical oxygen demand removal under denitrifying conditions.

Azoarcus↗
PubMed2001

The SBASE protein domain library, release 8.0: a collection of annotated protein sequence segments.

SBASE 8.0 is the eighth release of the SBASE library of protein domain sequences that contains 294 898 annotated structural, functional, ligand-binding and topogenic segments of proteins, cross-referenced to most major sequence databases and sequence pattern collections. The entries are clustered into over 2005 statistically validated domain groups (SBASE-A) and 595 non-validated groups (SBASE-B), provided with several WWW-based search and browsing facilities for online use. A domain-search facility was developed, based on non-parametric pattern recognition methods, including artificial neural networks. SBASE 8.0 is freely available by anonymous 'ftp' file transfer from ftp.icgeb.trieste.it. Automated searching of SBASE can be carried out with the WWW servers http://www.icgeb.trieste.it/sbase/ and http://sbase.abc. hu/sbase/.

Binding Sites↗
PubMed2004

Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs.

Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.

Bacteriophage P1↗
PubMed2004

Molecular cloning and endometrial expression of porcine high density lipoprotein receptor SR-BI during the estrous cycle and early pregnancy.

During rapid development of the fetus, levels of high density lipoprotein (HDL) are elevated in pregnant women. The receptor for HDL, scavenger receptor class B type I (SR-BI), mediates selective cholesteryl ester uptake and is highly expressed in the human placenta. Because of the rapid growth of uterus during early pregnancy and differences in placentation between swine and humans, we hypothesized that SR-BI may be expressed in porcine endometrium to take up HDL cholesterol. The objectives of this study were to obtain the full coding region for porcine SR-BI, determine endometrial expression of SR-BI mRNA during the estrous cycle and early pregnancy, and map the gene. By iterative screening of a porcine expressed sequence tag library, we obtained the full coding region of SR-BI. Endometrial expression of SR-BI in White composite gilts (n = 3-4 each) was determined by Northern blotting on Days 10, 13, and 15 cyclic gilts and Days 10, 13, 15, 20, 30, and 40 pregnant gilts. In cyclic gilts, endometrial expression of SR-BI did not change between Days 10 and 13, but increased (P < 0.01) between Days 13 and 15. In pregnant gilts, endometrial expression of SR-BI increased (P < 0.01) between Days 10 and 13, remained elevated until Day 30, and decreased (P = 0.015) on Day 40. The SR-BI gene was mapped to 46.3 cM on chromosome 14. These results show that endometrial expression of SR-BI changes during the estrous cycle and early pregnancy, and suggest that SR-BI takes up HDL for endometrial development during early pregnancy.

Amino Acid Sequence↗
PubMed2000

Cloning of the Atlantic salmon (Salmo salar) estrogen receptor-alpha gene.

A cDNA clone encoding most of an Atlantic salmon (Salmo salar) estrogen receptor (ER) was obtained from a liver cDNA library and the remainder of the coding sequence from the gene was isolated from a genomic library. Sequence comparisons showed that the cloned gene represents ER-alpha. Expression of the ER-alpha gene in male and female salmon parr was analysed by RT-PCR. Highest expression was found in brain and liver, with lower levels of ER-alpha mRNA present in all other tissues tested. There was little difference in expression of ER-alpha between male and female.

Amino Acid Sequence↗
PubMed2005

Comparison of ileum microflora of pigs fed corn-, wheat-, or barley-based diets by chaperonin-60 sequencing and quantitative PCR.

We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

Animal Feed↗
PubMed2003

An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling.

DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.

Adenosine Triphosphate↗
PubMed2006

Short polybasic peptide sequences are potent inhibitors of PC5/6 and PC7: Use of positional scanning-synthetic peptide combinatorial libraries as a tool for the optimization of inhibitory sequences.

Positional scanning-synthetic peptide combinatorial libraries (PS-SPCLs) are powerful molecular tools to identify enzyme substrate and potent inhibitory sequences and also to provide crucial information about active site determinants. PS-SPCLs have been surveyed for furin, proprotein convertase (PC)2, PC1/3, and PACE4 and proven efficient to identify potent peptidyl inhibitors in the low nanomolar range for furin and PC1/3. We report herein the screenings of nonamidated and acetylated hexapeptide PS-SPCLs for PC5/6A and PC7. The L-configuration library surveys distinctively revealed that L-Arg, L-Lys, and sometimes L-His in all six positions would generate the most potent inhibitors for both enzymes. Based on this clear polybasic preference, L-poly-Arg peptides ranging from four to nine residues were assayed. Inhibitory potency of these polybasic peptides increased with chain length, making nona-l-Arg a potent nanomolar inhibitor of PC5/6A and PC7 (Ki of 150 and 120 nM). PC5/6 and PC7 inhibition by nona-l-arginine was equivalent to that of furin (Ki of 114 nM) (J Biol Chem 275: 36741-36749, 2000). Nona-d-arginine was a more potent inhibitor of PC5/6 and PC7 than its levorotatory version (Ki of 19 and 81 nM), reminiscent of furin (Ki of 1.3 nM) (J Biol Chem 279:36788-36794, 2004). Our data indicate that certain poly-arginine peptides represent potent inhibitors targeting PCs of the constitutive secretory pathway (furin, PC5/6, and PC7). We conclude that basic residues within PC peptide inhibitors might be responsible for targeting PCs in general and for inhibitory potency, but that select amino acid changes will be necessary to acquire true specificity toward a single PC.

Amino Acid Sequence↗
PubMed2002

Characterization of porcine uterine estrogen sulfotransferase.

A quantitative trait locus (QTL) for uterine capacity is located on chromosome 8. Comparison of porcine and human genetic maps suggested that the estrogen sulfotransferase (STE) gene may be located near this region. The objectives of this study were to clone the full coding region for STE, compare endometrial STE gene expression between Meishan and White composite pigs during early pregnancy, and map the STE gene. We obtained a clone (1886 bp) containing the full coding region of STE by iterative screening of an expressed sequence tag library. Endometrial STE mRNA expression in White composite gilts was determined by Northern blotting on days 10, 13, and 15 of the estrous cycle; and on days 10, 13, 15, 20, 30, and 40 of pregnancy. STE mRNA expression was elevated (P < 0.01) on days 20 and 30 of pregnancy compared to other days of the cycle or pregnancy. Endometrial STE mRNA expression during early pregnancy, determined using real-time RT-PCR, was elevated (P < 0.01) on day 20 compared to day 15, decreased (P = 0.02) between days 20 and 30, and decreased further (P < 0.01) between days 30 and 40 in both Meishan and White composite pigs. Expression of STE mRNA was greater (P = 0.01) in White composite pigs compared to Meishan pigs. Using a microsatellite from an STE containing BAC genomic clone, the STE gene was mapped to 65 centimorgans on chromosome 8. Because STE mRNA expression differs between Meishan and White composite pigs, the STE gene may be a candidate for the uterine capacity QTL.

Amino Acid Sequence↗
PubMed2003

Molecular characterization and expression of porcine bone morphogenetic protein receptor-IB in the uterus of cyclic and pregnant gilts.

Previous gene mapping analyses revealed a quantitative trait locus for uterine capacity on chromosome 8. Comparison of porcine and human genetic maps suggests that the bone morphogenetic protein receptor IB (BMPR-IB) gene may be located near this region. The objectives of this study were to 1) clone the full coding region for BMPR-IB, 2) examine BMPR-IB gene expression by the endometrium and its cellular localization in cyclic and pregnant gilts, and 3) map the BMPR-IB gene. By iterative screening of an expressed sequence tag library, we obtained a 3559-base pair cDNA clone including the full coding region of BMPR-IB. Endometrial BMPR-IB mRNA expression of White composite gilts was determined by Northern blotting in Days 10, 13, and 15 cyclic and Days 10, 13, 15, 20, 30, and 40 pregnant gilts. In cyclic gilts, endometrial BMPR-IB mRNA expression was elevated on Days 13 and 15 (P < 0.01) compared with Day 10. Expression of BMPR-IB mRNA was localized in both luminal and glandular epithelium on Day 15. However, in pregnant gilts, BMPR-IB mRNA expression was not significantly different in the endometrium from Day 10 to Day 20, and it was significantly decreased on Days 30 and 40 (P = 0.011). The BMPR-IB gene was mapped to 108 cM on chromosome 8. These findings show that BMPR-IB mRNA expression is regulated differently in cyclic and pregnant gilts; this pattern of gene expression may be important for endometrial function during the luteal phase of the estrous cycle as compared with early pregnancy.

Amino Acid Sequence↗
PubMed2003

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants.

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.

Amino Acid Sequence↗
PubMed1993

Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library.

A human infant brain cDNA library, made specifically for production of expressed sequence tags (ESTs) was evaluated by partial sequencing of over 1,600 clones. Advantages of this library, constructed for EST sequencing, include the use of directional cloning, size selection, very low numbers of mitochondrial and ribosomal transcripts, short polyA tails, few non-recombinants and a broad representation of transcripts. 37% of the clones were identified, based on matches to over 320 different genes in the public databases. Of these, two proteins similar to the Alzheimer's disease amyloid precursor protein were identified.

Amino Acid Sequence↗