المؤلفون: Yiqun Hui, David J. Figueroa, Guochang Yang, Christopher P. Austin, Helen Galczenski, Neal G. Copeland, Colin D. Funk, Nancy A. Jenkins, Debra J. Gilbert

المصدر: Journal of Biological Chemistry. 276:47489-47495

مصطلحات موضوعية: Leukotriene, Leukotriene C4, Alternative splicing, HEK 293 cells, Cell Biology, respiratory system, Molecular cloning, Biology, Biochemistry, Molecular biology, chemistry.chemical_compound, Exon, chemistry, Complementary DNA, Coding region, lipids (amino acids, peptides, and proteins), Molecular Biology

الوصف: Two classes of cysteinyl leukotriene receptor, CysLT1 and CysLT2, have been identified and pharmacologically characterized in human tissues. Although the CysLT1 receptor mediates the proinflammatory effects of leukotrienes in human asthma, the physiological roles of CysLT2 receptor are not defined, and a suitable mouse model would be useful in delineating function. We report here the molecular cloning and characterization of the mouse CysLT2 receptor (mCysLT2R) from heart tissue. mCysLT2R cDNA encodes a protein of 309 amino acids, truncated at both ends compared with the human ortholog (hCysLT2R). The gene resides on the central region of mouse chromosome 14 and is composed of 6 exons with the entire coding region located in the last exon. Two 5′-untranslated region splice variants were identified with the short form lacking exon 3 as the predominant transcript. Although the overall expression of mCysLT2R is very low, the highest expression was detected in spleen, thymus, and adrenal gland by ribonuclease protection assay, and discrete sites of expression in heart were observed by in situ hybridization. Intracellular calcium mobilization in response to cysteinyl leukotriene administration was detected in human embryonic kidney 293T cells transfected with recombinant mCysLT2R with a rank order of potency leukotriene C4 (LTC4) = LTD4 ≫ LTE4. [3H]LTD4 binding to membranes expressing mCysLT2R could be effectively competed by LTC4 and LTD4 and only partially inhibited by LTE4 and BAYu9773. The identification of mCysLT2R will be useful for establishing CysLT2R-deficient mice and determining novel leukotriene functions.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::bff1ceeb092d4b44738bc4a20541b74aTest
https://doi.org/10.1074/jbc.m107556200Test

المؤلفون: Akira Okano, Takashi Furihata, Hiroyuki Nawa, Weidong Li, Tatsuo Suzuki, Qing-Bao Tian, Kohzo Nakayama

المصدر: Journal of Biological Chemistry. 276:21417-21424

مصطلحات موضوعية: Gene isoform, Aging, Transcription, Genetic, Molecular Sequence Data, Nerve Tissue Proteins, Biology, Biochemistry, Complementary DNA, Animals, Protein Isoforms, Coding region, Amino Acid Sequence, RNA, Messenger, Cloning, Molecular, Molecular Biology, Peptide sequence, Cells, Cultured, G alpha subunit, Cerebral Cortex, Neurons, chemistry.chemical_classification, Binding Sites, Reverse Transcriptase Polymerase Chain Reaction, cDNA library, GTPase-Activating Proteins, Alternative splicing, Gene Expression Regulation, Developmental, Genetic Variation, Cell Biology, Molecular biology, Rats, Amino acid, Alternative Splicing, chemistry

الوصف: We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e4ef1ec4dbecb32cd76f9e90ca02e862Test
https://doi.org/10.1074/jbc.m010744200Test

المؤلفون: Robert L. Medcalf, Marcus Tierney

المصدر: Journal of Biological Chemistry. 276:13675-13684

مصطلحات موضوعية: Messenger RNA, Exon, Exon trapping, Complementary DNA, Gene expression, Coding region, Cell Biology, Binding site, Biology, Molecular Biology, Biochemistry, Plasminogen activator, Molecular biology

الوصف: Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that inhibits urokinase. Constitutive and regulated PAI-2 gene expression involves post-transcriptional events, and an AU-rich mRNA instability motif within the 3′-untranslated region of PAI-2 mRNA is required for this process (Maurer, F., Tierney, M., and Medcalf, R. L. (1999)Nucleic Acids Res. 27, 1664–1673). Here we show that instability determinants are present within various exons of the PAI-2 coding region, most notably within exon 4. Deletion of exon 4 from the full-length PAI-2 cDNA results in a doubling in the half-life of PAI-2 mRNA, whereas a 28-nucleotide region within exon 4 contains binding sites for cytoplasmic proteins. Inducible stabilization of PAI-2 mRNA in HT-1080 cells treated with phorbol ester and tumor necrosis factor does not alter the binding of proteins to the exon 4 instability determinant, but resulted in a transient increase in the binding of factors to the AU-rich RNA instability element. Hence, PAI-2 mRNA stability is influenced by elements located within both the coding region and the 3′-untranslated region and that cytoplasmic mRNA binding factors may influence steady state and inducible PAI-2 mRNA expression. Finally a 10-nucleotide region flanking the exon 4 protein-binding site is homologous to instability elements within five other transcripts, suggesting that a common coding region determinant may exist.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::08bc76ba82b4b5bd6113f3e79801ea2eTest
https://doi.org/10.1074/jbc.m010627200Test

المؤلفون: Gerard Merkx, Tilo Schwientek, Henrik Clausen, Jiunn-Chern Yeh, Minoru Fukuda, Birgit Keck, Steven B. Levery, Ad Geurts van Kessel

المصدر: Journal of Biological Chemistry. 274:4504-4512

مصطلحات موضوعية: Expressed sequence tag, Chinese hamster ovary cell, Cellular differentiation, Cell Biology, Molecular cloning, Biology, Biochemistry, Molecular biology, Transmembrane protein, Gene product, Exon, Complementary DNA, Gene family, Coding region, Leukosialin, Gene, Molecular Biology, Peptide sequence

الوصف: A novel human UDP-GlcNAc:Gal/GlcNAcbeta1-3GalNAcalpha beta1, 6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-alpha-D-glucosamine:acceptor beta1, 6-N-acetylglucosaminyltransferase (beta1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2 O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a beta1,6GlcNAc-transferase that functions in both core 2 and core 4 O-glycan branch formation. The redundancy in beta1,6GlcNAc-transferases capable of forming core 2 O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::653f238375a60b25abe861fccc2a9fadTest
https://doi.org/10.1074/jbc.274.8.4504Test

المؤلفون: Minoru Fukuda, Edgar O. Ong, Jiunn-Chern Yeh

المصدر: Journal of Biological Chemistry. 274:3215-3221

مصطلحات موضوعية: DNA, Complementary, Surface Properties, Molecular Sequence Data, CHO Cells, Molecular cloning, N-Acetylglucosaminyltransferases, Biochemistry, Rapid amplification of cDNA ends, Cricetinae, Complementary DNA, Glycogen branching enzyme, Animals, Humans, Coding region, Amino Acid Sequence, Northern blot, Cloning, Molecular, Molecular Biology, Peptide sequence, Expressed sequence tag, Base Sequence, Sequence Homology, Amino Acid, biology, Mucins, Cell Biology, Molecular biology, biology.protein

الوصف: Mucin-type O-glycans are classified according to their core structures. Among them, cores 2 and 4 are important for having N-acetyllactosamine side chains, which can be further modified to express various functional oligosaccharides. Previously, we discovered by cloning cDNAs that the core 2 branching enzyme, termed core 2 beta-1,6-N-acetylglucosaminyltransferase-leukocyte type (C2GnT-L), is highly homologous to the I branching beta-1, 6-N-acetylglucosaminyltransferase (IGnT) (Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev. 7, 468-478). Using these homologous sequences as probes, we identified an expressed sequence tag in dbEST, which has significant homology to C2GnT-L and IGnT. This approach, together with 5'and 3' rapid amplification of cDNA ends, yielded a human cDNA that encompasses a whole coding region of an enzyme, termed C2GnT-mucin type (C2GnT-M). C2GnT-M has 48.2 and 33.8% identity with C2GnT-L and IGnT at the amino acid levels. The expression of C2GnT-M cDNA directed the expression of core 2 branched oligosaccharides and I antigen on the cell surface. Moreover, a soluble chimeric C2GnT-M had core 4 branching activity in addition to core 2 and I branching activities. A soluble chimeric C2GnT-L, in contrast, almost exclusively contains core 2 branching activity. Northern blot analysis demonstrated that the C2GnT-M transcripts are heavily expressed in colon, small intestine, trachea, and stomach, where mucin is produced. In contrast, the transcripts of C2GnT-L were more widely detected, including the lymph node and bone marrow. These results indicate that the newly cloned C2GnT-M plays a critical role in O-glycan synthesis in mucins and might have distinctly different roles in oligosaccharide ligand formation compared with C2GnT-L.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::73b8b517d1652273b108442d91211928Test
https://doi.org/10.1074/jbc.274.5.3215Test

المؤلفون: Eric P. Bennett, Henrik Clausen, Helle Hassan, Tilo Schwientek, Eric H. Holmes, Mitsuharu Nomoto, Margarida Amado, Raquel Almeida, Fátima Carneiro, Michael A. Hollingsworth, Steven B. Levery, Peter Aadal Nielsen

المصدر: Journal of Biological Chemistry. 273:12770-12778

مصطلحات موضوعية: Galactosyltransferase, Messenger RNA, Expressed sequence tag, Cell Biology, Biology, Biochemistry, Molecular biology, chemistry.chemical_compound, Exon, chemistry, Galactosamine, Complementary DNA, Coding region, Molecular Biology, Gene

الوصف: BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyltransferase, designated β3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to β3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyltransferase (β3Gal-T2) with similar kinetic properties as β3Gal-T1. Another gene represented a UDP-galactose:β-N-acetyl-galactosamine β-1,3-galactosyltransferase (β3Gal-T4) involved in GM1/GD1 ganglioside synthesis, and this gene was highly similar to a recently reported rat GD1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794–24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. β3Gal-T1 mRNA was expressed in brain, β3Gal-T2 was expressed in brain and heart, and β3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. β3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous β3-galactosyltransferase genes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::ed6cd605a07b5db1d65ff28ee002afafTest
https://doi.org/10.1074/jbc.273.21.12770Test

المؤلفون: Gary W. Conrad, Jennifer J. Swiergiel, Winston W.-Y. Kao, Atsushi Shiraishi, Richard Converse, Mary R. Roth, Saixia Ying, Candace W.-C. Kao, James L. Funderburgh

المصدر: Journal of Biological Chemistry. 272:30306-30313

مصطلحات موضوعية: Lumican, Transcription, Genetic, Keratan sulfate, Molecular Sequence Data, Restriction Mapping, Gene Expression, In situ hybridization, Biology, Biochemistry, Mice, Exon, chemistry.chemical_compound, Complementary DNA, Animals, Coding region, Tissue Distribution, Amino Acid Sequence, RNA, Messenger, Molecular Biology, Gene, In Situ Hybridization, Southern blot, Sulfates, Cell Biology, Molecular biology, Chondroitin Sulfate Proteoglycans, Genes, chemistry, Connective Tissue, Keratan Sulfate, sense organs

الوصف: Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3' noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5'-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a7ebc385e272765c01aca3bb8927733cTest
https://doi.org/10.1074/jbc.272.48.30306Test

المؤلفون: Michael P. Fautsch, Monique M. Perdok, Eric D. Wieben

المصدر: Journal of Biological Chemistry. 272:24691-24695

مصطلحات موضوعية: Untranslated region, Messenger RNA, Open reading frame, TATA box, Complementary DNA, CAAT box, Coding region, Cell Biology, Biology, Molecular Biology, Biochemistry, Gene, Molecular biology

الوصف: The GP1G gene of the guinea pig codes for three of the four abundant seminal vesicle secretory proteins produced in this species. This gene is expressed at highest efficiency in the seminal vesicle (SV) from a promoter that contains a canonical TATA box and CCAAT box. However, GP1G gene transcripts and proteins have also been identified in other tissues. To investigate the structure of GP1G transcripts produced in the testis, cDNA clones were isolated by screening a testis library. Three unique cDNAs (TSM1-3) were isolated. Each of these clones contained a 3'-untranslated region (UTR) and coding region identical to that of the seminal vesicle transcript. However, the 5'-UTRs of the testis transcripts were significantly longer than that found on the SV mRNA (416-646 nucleotides compared with only 23 nucleotides for the SV). Each of these alternatively spliced 5'-UTRs incorporated the SV promoter elements into transcribed sequence, and each contained multiple upstream AUG codons predicted to abolish translation of the major open reading frame. Nevertheless, each of the testis transcripts was capable of directing the synthesis of GP1G-related proteins in vitro. Analysis of the translation products suggests that the extended 5'-UTR of the testis transcripts regulate both the choice of translation start site and the efficiency of translation in this system. Western blot analysis of testis proteins revealed that the protein products of GP1G are also synthesized by the testis in vivo.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::03fa8503bf1de5d03efd61301f7620f6Test
https://doi.org/10.1074/jbc.272.39.24691Test

المؤلفون: Patrick Lau, Elizabeth T. Gum, Young S. Kim, James R. Gum, Samuel B. Ho, Neil W. Toribara

المصدر: Journal of Biological Chemistry. 272:16398-16403

مصطلحات موضوعية: chemistry.chemical_classification, Base pair, Cell Biology, Biology, Biochemistry, Molecular biology, Homology (biology), Amino acid, Rapid amplification of cDNA ends, chemistry, GenBank, Complementary DNA, Coding region, Molecular Biology, Peptide sequence

الوصف: The distribution of MUC6 suggests that its primary function is protection of vulnerable epithelial surfaces from damaging effects of constant exposure to a wide range of endogenous caustic or proteolytic agents. A combination of genomic, cDNA. and 3′ rapid amplification of cDNA ends techniques was used to isolate the carboxyl-terminal end of MUC6. The 3′ nontandem repeat region contained 1083 base pairs of coding sequence (361 amino acids) followed by 632 base pairs of 3′-untranslated region. The coding sequence consists of two distinct regions; region 1 contained the initial 270 amino acids (62% Ser-Thr-Pro with no Cys residues), and region 2 contained the COOH-terminal 91 amino acids (22% Ser-Thr-Pro with 12% Cys). Although region 1 had no homology to any sequences in GenBank, region 2 had approximately 25% amino acid homology to the COOH-terminal regions of human mucins MUC2, -5, and -5B and von Willebrand factor. The shortness of region 2 would leave little of the peptide backbone exposed to a potentially hostile environment. Antibody studies suggest that MUC6 in its native form exists as a disulfide-bonded multimer. The conservation of the 11 cysteine positions in region 2 suggests the importance of this short region to mucin polymerization.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::621311ee543414fbc278f375f7996b89Test
https://doi.org/10.1074/jbc.272.26.16398Test

المؤلفون: Shinya Yamashita, Chisako Segawa, Jun-ichiro Hata, Jiro Takeo

المصدر: Journal of Biological Chemistry. 272:7285-7289

مصطلحات موضوعية: endocrine system, DNA, Complementary, animal structures, animal diseases, Molecular Sequence Data, Biology, Biochemistry, chemistry.chemical_compound, Eukaryotic translation, Start codon, Complementary DNA, Protein biosynthesis, Animals, Humans, Coding region, Amino Acid Sequence, Cloning, Molecular, Peptide Chain Initiation, Translational, Molecular Biology, Methionine, Cell Biology, Molecular biology, Stop codon, Cell biology, chemistry, Oncorhynchus mykiss, Protein Biosynthesis, Fibroblast Growth Factor 2, Rainbow trout, Sequence Alignment

الوصف: Here, we describe the isolation of a rainbow trout cDNA clone that contains the entire fibroblast growth factor-2 (FGF-2; basic FGF) coding region. Interestingly, the rainbow trout cDNA contains a translation stop codon just upstream of the primary initiating methionine codon and so cannot give rise to the longer forms of FGF-2 that are produced in mammals by alternative translation initiation at leucines farther upstream. Transfection of human FGF-2 cDNA into fish cells shows that fish cells can initiate protein synthesis at an upstream leucine CUG codon; surprisingly, however, synthesis is initiated only at the most 5′ CUG and not at the two subsequent CUG codons or the methionine AUG codon also used in mammalian cells. Like other FGF-2 proteins, bacterially produced rainbow trout FGF-2 protein binds tightly to heparin-Sepharose and also promotes the proliferation of fibroblast cells. However, the protein differs from all others previously identified at amino acids 121-123, which are part of the proposed highly conserved receptor-binding domain. Comparisons of the efficacies of recombinant wild-type and mutant rainbow trout FGF-2 proteins demonstrate that these three amino acids are critical to the activity of FGF-2.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9d5b4ee5f54c7c8c9dd76b90d8c2c308Test
https://doi.org/10.1074/jbc.272.11.7285Test