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At least 685 records · Page 38Linked to original sources
PubMed2000

cDNA cloning of acyl-CoA desaturase homologs in the silkworm, Bombyx mori.

We have isolated two acyl-CoA desaturase clones from a pheromone gland cDNA library by using the EST (expressed sequence tag) database of Bombyx mori. The putative acyl-CoA desaturases encoded by the clones desat 1 (2029bp) and desat 2 (2341bp) have 98% identity, and both proteins show 61% identities to Trichoplusia ni acyl-CoA Delta(11) desaturase. The deduced amino acid sequences conserve well the histidine clusters that are catalytically essential for acyl-CoA desaturase activity. Northern blot and RT-PCR analyses revealed that both transcripts of desat 1 and desat 2 were expressed predominantly in the pheromone gland. Both transcripts detected 3days before adult eclosion dramatically increased a day before adult eclosion, keeping the mRNA levels high even after eclosion. These results, combined with the fact that Delta(11) and Delta(10, 12) desaturation of palmitate is a key step to synthesize pheromone in B. mori, suggest that the desaturases encoded by desat 1 and desat 2 are involved in either or both of the desaturation steps in the pheromone biosynthetic pathway of B. mori. The mRNA levels of desat 1 and desat 2 were not affected by decapitation or injection of the pheromone biosynthesis activating neuropeptide (PBAN) into the adult female moth, suggesting that the transcription of desat 1 and desat 2 is not regulated by PBAN. In addition to the clones in the pheromone gland, eight other clones encoding the same Delta(9) desaturase homolog were found in an embryonic cDNA library by searching from the EST database of B. mori. The deduced amino acid sequence from one of the clones (desat 3) shows 79% identity to T. ni Delta(9) desaturase but only 52% identity to the desaturases in the pheromone gland of B. mori. Northern blot analysis showed that the mRNA corresponding to the desat 3 was detected in the ovary and fat body, but not in the pheromone gland. Abundance of the Delta(9) desaturase clones (eight out of the 762 randomly sequenced clones) in the library prepared from diapause-destined embryos (40h after oviposition) suggests that the Delta(9) desaturase encoded by desat 3 plays an important role in embryonic development in B. mori.

Amino Acid Sequence↗
PubMed1996

Cloning and functional analysis of a beta-tubulin gene from a benomyl resistant mutant of Aspergillus parasiticus.

A genomic DNA library prepared from a benomyl resistant strain of Aspergillus parasiticus was screened with a Neurospora crassa beta-tubulin gene probe. A unique A. parasiticus genomic DNA fragment, thought to carry a mutant beta-tubulin gene (benr), was isolated. Two plasmids, pYT1 and pYTPYRG, carrying the putative benr gene or benr plus a second selectable marker (pyrG), respectively, were used to transform a benomyl sensitive strain of A. parasiticus (CS10) to determine if benr conferred benomyl resistance (BenR). BenR colonies were obtained with pYTPYRG, pYT1 or pYT1 cotransformed with pPG3J which carries a functional pyrG gene. No BenR colonies were obtained without added DNA or with pPG3J only (controls). Southern hybridization analysis of BenR and BenS transformants suggested that plasmid integration occurred most frequently at the chromosomal bens locus, however evidence for gene conversion and heterologous recombination was also observed. The predicted amino acid sequence of benr displayed a high degree of identity (> 93%) with other fungal beta-tubulin genes which confer benomyl resistance. Sequence analysis together with the genetic data suggested that benr encodes a functional mutant beta-tubulin.

Aspergillus↗
PubMed1997

Molecular cloning and expression of the large subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves.

A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley (Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley.

Amino Acid Sequence↗
PubMed1997

Isolation and expression pattern of hahr1, a homeobox-containing cDNA from Helianthus annuus.

A 2.5 kb homeobox (HB)-containing cDNA (hahr1) was isolated from a library prepared from rootlets of Helianthus annuus using a polymerase chain reaction (PCR)-based strategy. The putative protein product (77 kDa) contains the homeodomain (HD) and an acidic domain at the N-terminal region (residues 72-155). The deduced amino acid sequence of hahr1 shares a 53% sequence identity with GLABRA2, a HD protein associated with epidermal cell differentiation. Hahr1 expression was primarily found in dry seeds, hypocotyls and roots at stages associated with early developmental events. Expression was completely lacking in leaves and flowers. Evidence for the existence of one related gene expressed in sunflower stems was obtained by the presence of restriction fragment length polymorphism of amplified cDNA products.

Amino Acid Sequence↗
PubMed1998

Psx, a novel murine homeobox gene expressed in placenta.

To identify homeobox genes involved in mouse embryo development, we have screened a mouse cDNA library prepared from the 13.5-day-old entire conceptus for homeobox-containing sequences using a polymerase chain reaction (PCR) cloning strategy. We identified a mouse homeobox gene that might be the prototype of a new class. The full-length cDNA, designated Psx (placenta specific-homeobox), encodes a protein of 247 amino acids. The expression patterns of the Psx during embryonic development and in adult tissues were studied by Northern blot analysis. The Psx transcript was first detected at embryonic day 8.5 of conceptus and persisted until birth. Psx mRNA is expressed in extraembryonic tissues, mainly in placenta, but not in fetus, pups and adult tissues tested, suggesting that Psx plays an important role in placenta. Psx is a new member of the murine homeobox genes which are expressed in extraembryonic tissues such as placenta and amnion.

Amino Acid Sequence↗
PubMed1999

House-fly cytochrome P450 CYP6D1: 5' flanking sequences and comparison of alleles.

CYP6D1 is a cytochrome P450 found in the house fly, Musca domestica. Expression is greater in pyrethroid-resistant vs. -susceptible strains and can be induced by phenobarbital in adult susceptible flies. CYP6D1 is expressed only in adult flies. To gain information about possible regulatory elements involved in CYP6D1 expression, and to confirm the gene sequence that was previously determined by polymerase chain reaction (PCR), we screened a house-fly library prepared with genomic DNA from the pyrethroid-resistant LPR strain. A CYP6D1v1 clone was isolated and sequenced. This clone contained 887 nucleotides 5' to the open reading frame and a previously unknown 2.4-kb intron. Using polymerase chain reaction with primers based on the CYP6D1v1 allele, the sequences 5' to the ORF were obtained from five pyrethroid susceptible strains. The transcription initiation site (TIS) was identified at the same position in LPR and two susceptible strains (86 nucleotides upstream from the translation start site). A comparison of the 5' flanking sequences revealed a high degree of similarity for most regions, although differences in the sequences were identified. The possible roles of these sequence differences in regulation of CYP6D1 expression are discussed.

Alleles↗
PubMed1999

Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA.

A full-length cDNA for retinoblastoma (RB1) has been cloned from a cDNA library prepared from 3-week-old rainbow trout (Oncorhynchus mykiss) eyed embryos. The trout RB1 cDNA encodes a predicted protein of 910 amino acids and is the most divergent cloned retinoblastoma gene sequence to date. RT-PCR studies reveal high levels of RB1 expression by the second week of embryogenesis, which remains uniformly expressed until hatching. Expression studies of adult fish tissues show the RB1 gene to be expressed in all tissues examined, including the oesophagus, eye, liver, intestine, posterior and anterior kidney, skin, stomach, muscle, spleen, gill, swim bladder, gonads and brain. The RB1 gene appears to be a single copy gene based on Southern analysis, and maps to linkage group XVI in the trout genome map. Polymorphisms in the RB1 gene and in closely linked markers should facilitate LOH analysis of RB1.

Amino Acid Sequence↗
PubMed1999

Characterization of cDNAs encoding cytoplasmic ribosomal protein L15 and L27a in petunia (Petunia hybrida): primary structures and coordinate expression.

We have isolated two cDNA clones, PhRL15 and PhRL27a, encoding cytoplasmic ribosomal proteins L15 and L27a, from a cDNA library prepared from petunia petal protoplast cultures. An expressed sequence tag (EST) strategy was employed. PhRL15 and PhRL27a contained open reading frames corresponding to proteins of 204 and 150 amino acids and molecular weights of 24,100 and 17,000Da, respectively. The deduced amino acid sequences of these clones are about 60% to 70%, identical with those of yeast and rat. Southern blot analysis indicates that each gene may be encoded by a small multigene. The transcription levels of both clones were high in young vegetative organs, at the early event of floral development, and in dividing petal protoplast, and relatively low in highly differentiated reproductive organs. Our results demonstrate that the expression levels of both clones are correlated with the rate of growth and coordinatively controlled in petunia plant.

Amino Acid Sequence↗
PubMed1999

Characterization of katD, a kinesin-like protein gene specifically expressed in floral tissues of Arabidopsis thaliana.

Kinesin and kinesin-like proteins (KLPs) are microtubule-based motor proteins that play important roles in organelle transport. Based on the homology to these proteins, a katD cDNA has now been isolated from a library prepared from flowers of Arabidopsis thaliana ecotype Columbia. Sequence analysis of the katD cDNA revealed an open reading frame of 2691bp [corrected], encoding a protein of 987 amino acids. Comparison of the nucleotide sequences of katD genomic and cDNA clones revealed the presence of 18 introns, 17 of which conform to the GU-AG rule. The central region of the KatD polypeptide exhibits substantial amino acid sequence homology to the motor domain of kinesin heavy chains, although the motor domain of KatD appears to be phylogenetically distant from those of other KLPs in plants. The amino-terminal region of KatD shares marked sequence similarity with the calponin homology domain, whereas the approximately 240-residue carboxyl-terminal region shows no significant homology to other known proteins. The predicted secondary structure of KatD revealed the lack of an alpha-helical coiled coil structure typical of kinesin heavy chains, suggesting that KatD may function as a monomeric motor. A recombinant truncated KatD protein containing the putative motor domain was shown both to bind to mammalian microtubules in a manner dependent on a non-hydrolyzable ATP analog, and to possess microtubule-dependent ATPase activity. Immunoblot and Northern blot analyses showed that both KatD protein and mRNA are expressed specifically in floral tissues. These results suggest that the structurally distinct KatD protein functions as a floral tissue-specific motor protein.

Adenosine Triphosphatases↗
PubMed1999

Isolation and characterization of a cDNA clone encoding a putative white spruce glycine-rich RNA binding protein.

A presumably full-length cDNA encoding a putative glycine-rich RNA binding protein was isolated from a lambdaZAP cDNA library prepared from mRNAs extracted from needles of 2year old white spruce seedlings, which had been either wounded or jasmonate-treated. The cDNA, designated PgRNP (Picea glauca RNP protein), presents a 468bp open reading frame encoding a 155 amino acid protein. This polypeptide possesses an RNA binding domain (RNP-CS) and a glycine-rich domain. Comparative alignment reveals extensive homologies to glycine-rich RNA binding proteins containing an RNP-CS found in other angiosperm species. Genomic hybridization experiments suggest that the PgRNP gene is part of a small multigene family with at least four members. RNA blot analysis revealed that the PgRNP transcript is expressed in all tissues from non-stressed plants. Constitutive mRNA level was found in needle tissue from control as well as methyl-jasmonate treated plants. Wounding had no clear induction effect. Jasmonic acid treatment and systemic wound response had a positive effect on transcript accumulation. Transcript accumulation was slightly induced by cold in needles, and repressed by drought stress in both needle and root tissues of 2year old plants. Finally, the level of PgRNP accumulation was induced by wounding and repressed in 2week old dark-grown seedlings upon jasmonate treatments.

Acetates↗
PubMed2000

Xerl: a novel secretory protein expressed in eye and brain of Xenopus embryo.

A novel gene, Xerl (Xenopus EGF-like repeat with laminin-G domain protein) was isolated from a Xenopus head cDNA library prepared from tailbud. This gene encoded 779 amino acids including a potential signal sequence, twelve EGF-like repeats, a laminin-G domain, a RGD sequence and a VWF motif. In the EGF-like repeat and the laminin-G domain, Xerl showed similarity to those of Drosophila Crumbs, respectively. Zygotic expression of Xerl began at late gastrula, and increased through neurula up to the tailbud stage. In adult organs, Xerl was detected in brain and eye. Whole-mount in situ hybridization showed that Xerl expression occurred first in the anterior bilateral region of neurula and gradually localized to retina and forebrain and boundaries of midbrain and hindbrain.

Amino Acid Sequence↗
PubMed1997

Cloning of equine type II procollagen and the modulation of its expression in cultured equine articular chondrocytes.

The complete nucleotide sequence of equine type II procollagen has not been previously reported, and equine-specific probes have not been available. We report the complete sequence and discuss the molecular characteristics of equine type II procollagen mRNA which was cloned from a cDNA library prepared from mRNA isolated from equine articular chondrocytes. The coding sequence (4257 bp) was 92.4% homologous to the cDNA of the human sequence, and the propeptide was 97% identical to the human sequence. We demonstrated that when equine chondrocytes are grown in phenotypically-maintained cultures, the expression of type II procollagen is linked to the age of the animal. Additionally, in cultures of young equine chondrocytes, IL1-beta and TNF-alpha both reduced the expression of type II procollagen mRNA in a dose responsive manner.

Amino Acid Sequence↗
PubMed1993

Isolation of pMGT1: a gene that is repressed by oestrogen and increased by antioestrogens and antiprogestins.

In order to isolate additional markers of oestrogen responsiveness in breast cancer and to study the mechanisms associated with the development of endocrine resistance, we have searched for oestrogen regulated genes. Differential hybridisation analysis of a cDNA library prepared from oestrogen-stimulated T-47D cells has led to the isolation of a sequence (pMGT1) whose expression is repressed (up to 8-fold) by oestrogen (10(-9) mol/l) and represents the first down-regulated gene to be identified by this methodology. Further studies of pMGT1 expression in MCF-7 cells has revealed that the pure antioestrogens, ICI164384 (10(-7) mol/l) and ICI182780 (10(-7) mol/l) and the antiprogestin Ru38486 (10(-7) mol/l), increase pMGT1 mRNA levels by approximately 40-50-fold relative to the value seen in cells exposed to oestrogens. Under the same conditions, pS2(pLIV2), a gene which is positively regulated by oestradiol, was almost undetectable. Significantly, both tamoxifen (10(-7) mol/l), and 4-hydroxytamoxifen (10(-7) mol/l), failed to increase pMGT1 mRNA levels. Since cell culture studies have indicated that ICI164384 and ICI182780 are more effective than tamoxifen and 4-hydroxytamoxifen at inhibiting the growth of MCF-7 cells by mechanisms that lower their viability and sensitivity to growth factors, it is feasible that pMGT1 plays a central role in mediating these events and instigating pathways associated with cell death.

Breast Neoplasms↗
PubMed2003

2,6-disubstituted pyran-4-one and thiopyran-4-one inhibitors of DNA-Dependent protein kinase (DNA-PK).

6-aryl-2-morpholin-4-yl-4H-pyran-4-ones and 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones were synthesised and evaluated as potential inhibitors of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). Several compounds in each series exhibited superior activity to the chromenone LY294002, and were of comparable potency to the benzochromenone NU7026 (IC(50)=0.23 microM). Importantly, members of both structural classes were found to be selective inhibitors of DNA-PK over related phosphatidylinositol 3-kinase-related kinase (PIKK) family members. A multiple-parallel synthesis approach, employing Suzuki cross-coupling methodology, was utilised to prepare libraries of thiopyran-4-ones with a range of aromatic groups at the 3'- and 4'-positions on the thiopyran-4-one 6-aryl ring. Screening of the libraries resulted in the identification of 6-aryl-2-morpholin-4-yl-4H-thiopyran-4-ones bearing naphthyl or benzo[b]thienyl substituents at the 4'-position, as potent DNA-PK inhibitors with IC(50) values in the 0.2-0.4 microM range.

Animals↗
PubMed2002

cDNA sequences and mRNA levels of two hexamerin storage proteins PinSP1 and PinSP2 from the Indianmeal moth, Plodia interpunctella.

In insects, storage proteins or hexamerins accumulate apparently to serve as sources of amino acids during metamorphosis and reproduction. Two storage protein-like cDNAs obtained from a cDNA library prepared from fourth instar larvae of the Indianmeal moth (Plodia interpunctella) were cloned and sequenced. The first clone, PinSP1, contained 2431 nucleotides with a 2295 nucleotide open reading frame (ORF) encoding a protein with 765 amino acid residues. The second cDNA, PinSP2, consisted of 2336 nucleotides with a 2250-nucleotide ORF encoding a protein with 750 amino acid residues. PinSP1 and PinSP2 shared 59% nucleotide sequence identity and 44% deduced amino acid sequence identity. A 17-amino acid signal peptide and a molecular mass of 90.4 kDa were predicted for the PinSP1 protein, whereas a 15-amino acid signal peptide and a mass of 88 kDa were predicted for PinSP2. Both proteins contained conserved insect larval storage protein signature sequence patterns and were 60-70% identical to other lepidopteran larval storage proteins. Expression of mRNA for both larval storage proteins was determined using the quantitative reverse transcription polymerase chain reaction method. Only very low levels were present in the second instar, but both mRNAs dramatically increased during the third instar, peaked in the fourth instar, decreased dramatically late in the same instar and pupal stages, and were undetectable during the adult stage. Males and females exhibited similar mRNA expression levels for both storage proteins during the pupal and adult stages. The results support the hypothesis that P. interpunctella, a species that does not feed after the larval stage, accumulates these two storage proteins as reserves during larval development for subsequent use in the pupal and adult stages.

Amino Acid Sequence↗
PubMed2000

Molecular cloning and expression of CYP6B8: a xanthotoxin-inducible cytochrome P450 cDNA from Helicoverpa zea.

Xanthotoxin, a plant allelochemical, induces alpha-cypermethrin insecticide tolerance in Helicoverpa zea (corn earworm); inhibition of tolerance by piperonyl butoxide implicates cytochrome P450 monooxygenases (P450s) in the detoxification of this insecticide. To characterize the xanthotoxin-inducible P450 that might mediate alpha-cypermethrin tolerance in this species, a cDNA library prepared from xanthotoxin-induced H. zea fifth instar larvae was screened with cDNAs encoding furanocoumarin-metabolizing P450s from Papilio polyxenes (CYP6B1v2) and P. glaucus (CYP6B4v2) as well as a sequence-related P450 from Helicoverpa armigera (CYP6B2). One full-length cDNA isolated in this screening shares 51-99% amino acid identity with the CYP6B subfamily of P450s isolated from Papilio and Helicoverpa species and, thus, has been designated CYP6B8. All of these CYP6B subfamily members share a number of highly conserved domains, including substrate recognition site 1 (SRS 1) that is critical for xanthotoxin metabolism by CYP6B1v2 from Papilio polyxenes and coumarin metabolism by CYP2a5 from Mus musculus. Northern and RT-PCR analyses indicate that CYP6B8 expression is strongly induced by xanthotoxin and phenobarbital and negligibly induced by alpha-cypermethrin.

Amino Acid Sequence↗
PubMed2002

Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima.

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K(i) value of 0.06 microM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K(i) value of 1 microM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and functional expression of a cDNA encoding the major endoplasmic reticulum-associated calcium-binding protein, calreticulin, from Philippine strain Schistosoma japonicum.

We describe the cloning of a full length calreticulin (CR)-encoding cDNA clone isolated by immunoscreening of a cDNA library prepared with mRNA from adult worms of the Philippine strain of Schistosoma japonicum, the cause of Asian schistosomiasis. The sequence of the cDNA is presented, and its molecular characterisation and functional expression as a Ca2+-binding protein described. The potential role of CR in inducing protective immunity in the schistosomes is discussed.

Amino Acid Sequence↗