SEARCH · PubMed Health

Results for “Library preparation”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources

PubMed1998

Cloning and functional characterization of PACAP-specific receptors in zebrafish.

Two PACAP receptors were isolated from total zebrafish cDNA library prepared from 6-day old fish by a homology-based cloning strategy. The two zebrafish PACAP receptors have the same topology as the one found in other members of this class of seven membrane-spanning G-protein-coupled receptors. Each of the two zebrafish PACAP receptors shares about 70% sequence identity at the amino acid level with the human PACAP-type 1 receptor, and about 50% amino acid identity with PACAP/VIP R-1 and PACAP/VIP R-2 receptors. One of these zebrafish receptors contains the hop2 configuration found in the human and rat PACAP-type 1 receptors. On the basis of these structural characteristics the zebrafish PACAP receptors were classified as PACAP-type 1 and PACAP-type 2 receptors. In competitive binding experiments zebrafish PACAP-type 1 and PACAP-type 2 receptors showed similar binding specificity for zebrafish and human PACAP-38 and PACAP-27. Furthermore, the specificity of PACAP-type 1 and PACAP-type 2 receptors for zebrafish and human PACAPs is about 1,000-fold higher than for human VIP. These results demonstrate that zebrafish PACAP-type 1 receptor is a structural and pharmacological homolog of the mammalian PACAP-type 1 receptor. Additional pharmacological characterization is needed in order to classify the zebrafish PACAP-type 2 receptor.

Amino Acid Sequence↗

PubMed1997

Genes induced in programmed cell death of neuronal PC12 cells and developing sympathetic neurons in vivo.

To identify primary response genes induced during early stages of neuronal programmed cell death (PCD), we screened by differential hybridization a subtracted cDNA library prepared from neuronal PC12 cells deprived of NGF for 6 hr in the presence of cycloheximide. Eight induced cDNA sequences were identified and designated message up-regulated during death (mud)-1-8. To determine which cloned sequences might be involved in neuronal PCD in vivo, expression of mud genes was analyzed in developing rat superior cervical ganglia (SCG) undergoing programmed cell death, using a combination of reverse Southern, reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization. Five sequences (mud-1, -3, -5/8, -6, and -7) are induced in SCG undergoing cell death in vivo, and induction of at least three of these (mud-3, -6, and -7) occurs in neurons. Partial sequence analysis reveals that mud-1 corresponds to annexin VI; mud-3 corresponds to rat PC3, mouse TIS21; mud-4 appears to be the rat homolog of human TAFII70; mud-5 and -8 are >85% identical members of the rodent gene family of B2-transcribed repeats; and mud-6 appears to be the rat homolog of human Ring 3 and Drosophila female sterile homeotic (fsh). Mud-2 and mud-7 encode novel sequences. These new candidate genes provide markers for early stages of neuronal PCD, are potentially involved in the cell death process, and serve to expand our view of cell death control in the developing nervous system.

Animals↗

PubMed1992

Isolation and characterization of a cDNA that encodes ECP31, an embryogenic-cell protein from carrot.

A full-length cDNA for ECP31, an embryogenic cell protein from carrot (Daucus carota L.) with a M(r) of 31,000 (Kiyosue T, Satoh S, Kamada H, Harada H (1991) Plant Physiol 95: 1077-1083), was isolated from a cDNA library prepared from embryogenic cells using PCR-amplified DNA as a probe. The genomic Southern blot analysis revealed that there are two or three genes for ECP31 in the carrot genome. The transcripts of ECP31 accumulated in the peripheral regions of clusters of embryogenic cells and disappeared in the course of somatic embryogenesis that was induced by transfer of the embryogenic cells to auxin-free media. The cDNA encodes a polypeptide of 256 amino acids, and the calculated molecular weight of this polypeptide is 26,111. The deduced amino acid sequence shows a high degree (62.2%) of similarity to that of a protein that is abundant during late embryogenesis of cotton (LEA D34; Baker JC, Steele C, Dure III (1988) Plant Mol Biol 11: 227-291). The mRNAs for ECP31 started to accumulate in zygotic embryos at a late stage of embryogenesis but were undetectable in mature embryos within 24 h after imbibition of seeds. In dry fruits (seeds), the transcripts were detected only in zygotic embryos by in situ hybridization. The level of ECP31 transcripts increased after treatment with abscisic acid (ABA) in torpedo-shaped somatic embryos but not in seven-day-old seedlings. These results suggest that both embryo-specific factor(s) and ABA are involved in the expression of the gene for ECP31.

Abscisic Acid↗

PubMed1999

Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, jug r 2, from english walnut kernel (Juglans regia), a major food allergen.

BACKGROUND: Walnuts and other tree nuts are important food-allergen sources that have the potential to be associated with life-threatening, IgE-mediated systemic reactions in some individuals. OBJECTIVE: The purpose of this study was to characterize a complementary (c)DNA clone encoding one of the walnut food allergens. METHODS: A cDNA expression library prepared from walnut somatic embryo was screened for IgE reactivity with patient serum. A reactive clone of 2060 bp, which encoded a protein of 593 amino acids in length, was subcloned by excision into the pGEX expression vector. IgE-binding inhibition experiments were performed. RESULTS: A recombinant fusion protein was induced and shown to bind serum IgE from 9 of 15 patients tested, thus identifying a major allergen. This clone, named Jug r 2, exhibited significant homology with genes encoding the vicilin group of seed proteins. An IgE-binding inhibition experiment suggested that the encoded protein undergoes posttranslational modification into at least one major polypeptide (47 kd) and possibly several others, which is similar to the vicilin-like proteins characterized in cocoa bean (Theobroma cacao) and cottonseed (Gossypium hirsutum). N-terminal sequencing of the 47-kd band, Jug r 2, identified it as a mature protein obtained from the precursor. A second IgE-binding inhibition experiment showed that there is minimal or no cross-reactivity between Jug r 2 and pea vicilin, peanut proteins, or cacao proteins. CONCLUSION: Jug r 2 is the third vicilin food allergen identified in addition to vicilins from soy and peanut. The availability of recombinant food allergens should help advance studies on the immunopathogenesis and possible treatment of IgE-mediated food hypersensitivity.

Allergens↗

PubMed2001

Molecular cloning and characterization of a novel Jasmonate inducible pathogenesis-related class 10 protein gene, JIOsPR10, from rice (Oryza sativa L.) seedling leaves.

A novel rice (Oryza sativa L.) gene, homologous to a sorghum pathogenesis-related class 10 protein gene, was cloned from a cDNA library prepared from 2-week-old jasmonic acid-treated rice seedling leaves, and named as JIOsPR10 (jasmonate inducible). JIOsPR10 encoded a 160-amino-acid polypeptide with a predicted molecular mass of 17,173.23 Da and a pI of 5.84. JIOsPR10 was highly similar (77%) to the sorghum PR10 protein, but showed less than 55% similarity with other identified PR10s at the amino acid level. Genomic Southern analyses indicated the presence of related genes in the rice genome. The JIOsPR10 transcript was not detected in the healthy leaves, and was not induced after cut. Further expression analysis revealed that the signaling components of defense/stress pathways, jasmonate, salicylate, and H(2)O(2) significantly up-regulated the JIOsPR10 mRNA over the cut control, whereas two other stress regulators, ethylene and abscisic acid, failed to induce its expression. Interestingly the protein phosphatase (PP) inhibitors, cantharidin, endothall, and okadaic acid, rapidly and potently up-regulated the JIOsPR10 expression, suggesting involvement of the phosphorylation/dephosphorylation events. Additionally, the inducible expression of the JIOsPR10 gene was influenced by light signal(s). Finally, the blast pathogen (Magnaporthe grisea) also specifically elicited the accumulation of JIOsPR10 mRNA in leaves. Induction of the JIOsPR10 gene expression by signaling molecules, PP inhibitors and pathogen attack, strongly indicate a role for this novel gene in rice self-defense/stress response(s).

Amino Acid Sequence↗

PubMed1997

Expression cloning of rat cDNA encoding UDP-galactose:GD2 beta1,3-galactosyltransferase that determines the expression of GD1b/GM1/GA1.

Using an anti-GD1b monoclonal antibody, expression cloning of a cDNA for the beta1,3-galactosyltransferase gene (EC 2.4.1.62) was performed. KF4C, mouse melanoma B16 transfected with polyoma T antigen gene, and GM2/GD2 synthase cDNA was used as a recipient cell line for the cDNA library transfection. A cDNA clone of GD3 synthase, pD3T-31 was co-transfected with a cDNA library prepared from rat brain RNA using the pcDNAI expression vector. The isolated cDNA clone pM1T-9 predicted a type II membrane protein with 4 amino acids of cytoplasmic domain, 21 amino acids of transmembrane region, and a large catalytic domain with 346 amino acids. Introduction of the cDNA clone into a mouse melanoma line B16 previously transfected with a GM2/GD2 synthase gene resulted in the neo-synthesis of GM1. Co-transfection of the cell line with pM1T-9 and a GD3 synthase cDNA resulted in the expression of GD1b as well as GM1. Moreover, introduction of pM1T-9 into L cell (lacking GM3 synthase), previously transfected with GM2/GD2 synthase gene, resulted in the definite expression of asialo-GM1. These results indicated that GD1b/GM1/GA1 synthases were identical, as previously suggested based on enzymological analysis. In Northern blots of the beta1, 3-galactosyltransferase gene with total RNA from various rat tissues, a 1.6-kilobase mRNA was strongly expressed in spleen, thymus, kidney, and testis. However, the expression level of the gene in the adult brain tissue was not especially high. On the other hand, this gene was expressed at high levels in the rat brain of embryonal day 12, and reached a peak at around birth, then fell to low level in the adult brain.

Aging↗

PubMed1991

Cloning and expression of the human vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator found in the central and peripheral nervous system. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guanine nucleotide-binding (G) protein capable of activating adenylate cyclase. A cDNA clone (GPRN1) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line of leukemic pre-B lymphoblasts and the HT-29 line of colon carcinoma cells. The deduced 362-amino acid polypeptide sequence encoded by GPRN1 shares a seven-transmembrane-segment hydropathicity profile with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kilobase transcript of the VIP receptor in Nalm 6 and HT-29 cells as well as in tissues from rat brain, colon, heart, lung, kidney, spleen, and small intestine. COS-6 cells transfected with GPRN1 bound 125I-labeled VIP specifically with a dissociation constant (Kd) of 2.5 nM. VIP--and less effectively secretin, peptide histidine isoleucine (PHI), and glucagon competitively displaced bound 125I-VIP from transfected COS-6 cells, with potencies in the order VIP greater than secretin = PHI much greater than glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3-fold increase in the intracellular level of cAMP. When the antisense orientation of the VIP receptor clone was introduced into HT-29 cells, there was a 50% suppression of the specific binding of 125I-VIP and of the VIP-induced increase in cAMP level, relative to untransfected cells. The VIP receptor cloned exhibits less than or equal to 24% homology with other receptors in the same superfamily and thus represents a subset of G protein-coupled receptors for peptide ligands.

Amino Acid Sequence↗

PubMed2000

Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos.

A cDNA library prepared from fertilized eggs of the ascidian Halocynthia roretzi was screened for prelocalized mRNAs in the early embryo by means of whole-mount in situ hybridization using a digoxigenin-labeled antisense RNA of each clone. Random mass screening of 150 cDNAs in a fertilized egg yielded six different clones which showed mRNA localization in the posterior-vegetal cytoplasm of the 8-cell embryo. An in situ hybridization study of the detailed spatial distribution of each mRNA in embryos of various stages revealed that there are, in contrast to the identical localization in embryos after the 16-cell stage, two distinct patterns of RNA distribution at earlier stages. One is colocalization with the myoplasm from the prefertilization stage to the 8-cell stage (type I postplasmic RNAs). The other is delayed accumulation of RNA at the posterior-vegetal cytoplasm after fertilization (type II postplasmic RNAs). We found that both types of RNAs associate with the cytoskeleton, but that they show different sensitivities to inhibitors of the cytoskeleton; translocation of the type I RNAs is dependent upon microfilaments during the first phase of ooplasmic segregation and dependent upon microtubules during the second phase of segregation, whereas translocation of the type II RNAs is dependent upon microfilaments throughout ooplasmic segregation. These results show that there are two pathways for the localization of the RNAs at the posterior-vegetal cytoplasm in the 8-cell embryo of the ascidian H. roretzi.

Amino Acid Sequence↗

PubMed2006

Characterization, cloning and expression of the ferritin gene from the Korean polychaete, Periserrula leucophryna.

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.

Amino Acid Sequence↗

PubMed1991

Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte "homing" receptor expressed by hemopoietic cells.

In addition to the 85-95 kD CD44 species found on most hemopoietic cell types, the human myelomonocytic cell line KG1a expresses proteins of approximately 115 kD and 130 kD that react with monoclonal antibodies belonging to CD44. The possibility that these higher molecular weight species may represent novel CD44 isoforms containing additional protein sequence was investigated. CD44 cDNA clones were isolated from a plasmid-based expression library prepared from KG1a mRNA. One of the three clones obtained (clone 2.3) was found to encode a CD44 molecule of approximately 130 kD in transfected COS cells. Sequences analysis indicated that the molecule encoded by this cDNA clone, designated CD44R1, was essentially identical to CD44 except for the presence of an additional 132 amino acids inserted into the extracellular domain. This inserted region is rich in serine and threonine residues that may serve as sites of O-linked glycosylation, and contains a potential site of N-linked glycosylation and a potential site of chondroitin sulphate attachment. PCR analysis using primers that flank the inserted region present within CD44R1 identified an additional CD44 isoform, designated CD44R2, that contains only the last 69 amino acids present within the unique region of CD44R1. Peripheral blood mononuclear cells and granulocytes from normal individuals and patients with chronic myelogenous leukemia, polycythemia vera, or acute myelomonocytic leukemia, express both CD44R1 and CD44R2. In contrast, CD44R1 and CD44R2 appear to be differentially expressed in various CD44-positive cell lines. Thus KG1a, and the Epstein-Barr Virus-transformed B cell lines WalkDR4 and Way-1 express both CD44 and the CD44 isoforms CD44R1 and CD44R2, while the myeloid cell lines HL60 and U937 express high levels of CD44, but only very low levels of CD44R1 and CD44R2. The CD44-negative cell lines DHL-4, DHL-10, Jurkat, and K562 are also negative for CD44R1 and CD44R2.

Amino Acid Sequence↗

PubMed2006

Alternaria alternata NADP-dependent mannitol dehydrogenase is an important fungal allergen.

BACKGROUND: Alternaria alternata is one of the most important allergenic fungi worldwide. Mannitol dehydrogenase (MtDH) has previously been shown to be a major allergen of Cladosporium herbarum and cross-reactivity has been demonstrated for several fungal allergens. OBJECTIVE: The present study's objective was to clone the MtDH from an A. alternata cDNA library, express and purify the recombinant non-fusion protein and test its IgE-binding properties. Methods A cDNA library prepared from A. alternata hyphae and spores was screened for mannitol dehydrogenase by DNA hybridization with the radioactively labelled C. herbarum homologue as a probe. The resulting clone was sequenced and heterologously expressed in Escherichia coli as a recombinant non-fusion protein, which was purified to homogeneity and analysed for its IgE-binding capacity. RESULTS: The coding sequence of the full-length cDNA clone comprises 798 bp encoding a protein with a molecular mass of 28.6 kDa and a predicted pI of 5.88. Protein sequence analysis revealed an identity of 75% and a homology of 86% between the MtDHs of A. alternata and C. herbarum. The functional mannitol dehydrogenase was expressed in the E. coli strain BL21(DE3) transformed with the vector pMW172 and purified to homogeneity. The enzyme catalyses the NADPH-dependent conversion of d-fructose to d-mannitol. In IgE-ELISA and immunoblots, MtDH is recognized by 41% of A. alternata-allergic patients. In vivo immunoreactivity of the recombinant MtDH was verified by skin prick testing. Finally, inhibition-ELISA experiments confirmed cross-reactivity between the MtDHs of A. alternata and C. herbarum. CONCLUSION: Mannitol dehydrogenase (Alt a 8) represents an important new allergen of the ascomycete A. alternata that might be suitable for improving diagnostic and therapeutic procedures.

Allergens↗

PubMed1990

The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19.

Amino Acid Sequence↗

PubMed2001

Genomic organization and differential expression of channel catfish MHC class I genes.

Two clones, designated Icpu-UA/3 and Icpu-UA/26, were isolated from a genomic library prepared from a single homozygous gynogenetic channel catfish. Sequence analysis showed that each clone encoded a gene product containing features conserved among MHC class I molecules. The genomic organization of both clones indicated that each domain, with the exception of the cytoplasmic, was encoded by a separate exon. Moreover, like mammals, catfish cytoplasmic regions were encoded by three exons rather than two as previously described for other teleost MHC class I genes. Analysis of nucleotide sequences upstream of catfish class I genes revealed the presence of several regulatory motifs similar to those seen in mammalian class I genes. These included a TATA box, Enhancer B, Site alpha, ISRE, and GAS elements. To determine the functional significance of these elements, EMSAs and tissue expression assays were performed. EMSAs demonstrated that an Enhancer B element within Icpu-UA/26, and an imperfect Enhancer B element and/or a GC-rich region within Icpu-UA/3 were responsible for formation of specific DNA/protein complexes. Expression studies detected Icpu-UA/26 transcripts in all tissues tested, whereas Icpu-UA/3 encoded messages were seen in a limited number of tissues. These results define the intron/exon organization of catfish MHC class I genes, suggest that Icpu-UA/3 encodes a nonclassical gene, and provide the first functional evidence that upstream sequences, similar to those seen in mammalian class I genes, play important roles in regulating teleost MHC gene expression.

Amino Acid Sequence↗

PubMed2001

Screening of environmental DNA libraries for the presence of genes conferring Na(+)(Li(+))/H(+) antiporter activity on Escherichia coli: characterization of the recovered genes and the corresponding gene products.

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na(+)(Li(+))/H(+) antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li(+)-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na(+)/H(+) antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na(+)/H(+) antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The K(m) values of both antiporters for Na(+) were approximately 10-fold higher than the values for Li(+).

Amino Acid Sequence↗

PubMed2002

Cloning, partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis.

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.

Animals↗

PubMed1992

cDNA clone to chick corneal chondroitin/dermatan sulfate proteoglycan reveals identity to decorin.

A 1.6-kb cDNA clone was isolated by screening a library prepared from chick corneal mRNA with a cDNA clone to bovine decorin. The cDNA contained an open reading frame coding for a M(r) 39,683 protein. A 19-amino-acid match with sequence from the N-terminus of core protein from the corneal chondroitin/dermatan sulfate proteoglycan confirmed the clone as a corneal proteoglycan and the homology with human and bovine decorin confirmed its identity as decorin. Structural features of the deduced sequence include a 16-amino-acid signal peptide, a 14-amino-acid propeptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 63% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I. Chick decorin contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The presence of beta bend amino acids flanking the units may serve to delineate the units as structural elements of the leucine-rich region. Sequence homology within the repeats and the spacing of the repeats suggest that this region arose by duplication. Chick decorin primarily differs from mammalian decorins in the 19-amino-acid sequence that starts the N-terminus of the core protein. Within this region, the serine that serves as a potential acceptor for the chondroitin/dermatan sulfate side chain is preceded by a glycine instead of being followed by a glycine as it is in the mammalian decorins and all other mammalian proteoglycans.

Amino Acid Sequence↗

PubMed1998

An epidermis/papilla-specific oxalate oxidase-like protein in the defence response of barley attacked by the powdery mildew fungus.

A cDNA clone of a defence response transcript was isolated from a library prepared from barley leaves expressing papilla resistance towards the powdery mildew fungus, Blumeria (syn. Erysiphe) graminis f.sp. hordei (Bgh). The 904 bp sequence encodes a 229 amino acid polypeptide with a putative signal peptide of 23 amino acids. After cleavage, the protein has a mass of 22.3 kDa and exhibits up to 60% amino acid identity to certain dicot proteins, and 46% amino acid identity to barley oxalate oxidase; therefore we designated it HvOxOLP (for Hordeum vulgare oxalate oxidase-like protein). Single-base substitutions among several cDNA and RACE clones demonstrate a gene of many copies. Both the transcript and protein accumulate from 3 h after inoculation with Bgh. The transcript level peaks at 18-24 h and subsequently decreases, whereas the protein level is stable from 24 h after inoculation. The accumulation patterns are independent of the outcome of the barley/powdery mildew interaction, unlike that of PR proteins, for example. The transcript accumulates specifically in the inoculated epidermal tissue. This temporal and spatial expression pattern suggests a very close relationship to papilla formation. Immunoblot analyses have facilitated a demonstration that HvOxOLP, like oxalate oxidase, is a water-soluble 100 kDa oligomeric protein. The oligomer is heat-stable and SDS-tolerant, and it can be denatured into a 25 kDa monomer. Attempts to demonstrate oxalate oxidase activity for this protein have failed. However, the relationships to oxalate oxidase suggests that HvOxOLP may be involved in H2O2 generation necessary for, for example, cross-linking of cell wall components during formation of papillae.

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence of the gene for the b subunit of human factor XIII.

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.

Amino Acid Sequence↗