Journal articles on the topic 'Human brain cDNA library'
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1
Adams, Mark D., M. Bento Soares, Anthony R. Kerlavage, Chris Fields, and J. Craig Venter. "Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library." Nature Genetics 4, no. 4 (August 1993): 373–80. http://dx.doi.org/10.1038/ng0893-373.
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Xu, Lei, Jingqi Li, Li Liu, Lixia Lu, Jingxia Gao, and Xueli Li. "Construction of equalized short hairpin RNA library from human brain cDNA." Journal of Biotechnology 128, no. 3 (February 20, 2007): 477–85. http://dx.doi.org/10.1016/j.jbiotec.2006.11.013.
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Nobis, William, Xiaoning Ren, Steven P. Suchyta, Thomas R. Suchyta, Adroaldo J. Zanella, and Paul M. Coussens. "Development of a porcine brain cDNA library, EST database, and microarray resource." Physiological Genomics 16, no. 1 (December 16, 2003): 153–59. http://dx.doi.org/10.1152/physiolgenomics.00099.2003.
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Recent developments in expressed sequence tag (EST) and cDNA microarray technology have had a dramatic impact on the ability of scientists to study responses of thousands of genes to internal and external stimuli. In neurobiology, studies of the human brain have been expanding rapidly by use of functional genomics techniques. To enhance these studies and allow use of a porcine brain model, a normalized porcine brain cDNA library (PBL) has been generated and used as a base for EST discovery and microarray generation. In this report, we discuss initial sequence analysis of 965 clones from this resource. Our data revealed that library normalization successfully reduced the number of clones representing highly abundant cDNA species and overall clone redundancy. Cluster analysis revealed over 800 unique cDNA species representing a redundancy rate for the normalized library of 6.9% compared with 29.4% before normalization. Sequence information, BLAST results, and TIGR cluster matches for these ESTs are publicly available via a web-accessible database ( http://nbfgc.msu.edu ). A cDNA microarray was created using 877 unique porcine brain EST amplicons spotted in triplicate on glass slides. This microarray was assessed by performing a series of experiments designed to test hybridization efficiency and false-positive rate. Our results indicate that the PBL cDNA microarray is a robust tool for studies of brain gene expression using swine as a model system.
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Gong, Yuewen, Manna Zhang, Li Cui, and Gerald Y. Minuk. "Sequence and chromosomal assignment of a human novel cDNA: similarity to gamma-aminobutyric acid transporter." Canadian Journal of Physiology and Pharmacology 79, no. 12 (December 1, 2001): 977–84. http://dx.doi.org/10.1139/y01-082.
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Gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the mammalian brain. Although initially thought to be confined to the central nervous system, GABAergic activity has also been described in other tissues throughout the body. In the present study, we report the cloning and localization of human GABA transporter cDNA and document its expression in various human tissues. A human liver cDNA library was initially screened by a 32P-labeled murine brain GABA transporter 3 (GAT-3) cDNA probe, and full-length cDNA was cloned by employing Marathon-Ready(tm) human kidney cDNA. The human GABA transporter cDNA encoded a 569 amino acid hydrophobic protein with 12 transmembrane domains (TMs). Search of published sequences revealed high homology with rat GAT-2, murine GAT-3 cDNA, human solute carrier family 6 member 13 (SLC6A13), and a human peripheral betaine/GABA transporter. Northern blot analyses demonstrated that the human GABA transporter is expressed strongly in the kidney and to a lesser extent in the liver and brain. The sequence was well matched with human chromosome 12p13.3, suggesting the human GABA transporter contains 14 exons. The above findings confirm the existence of and further characterize a specific GABA transporter in human tissues.Key words: sequence, chromosome, GABA, GABA transporter.
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MICHEL, UWE, BORIS KALLMANN, PETER RIECKMANN, and DIRK ISBRANDT. "UM 9(5)h and UM 9(5)p, human and porcine noncoding transcripts with preferential expression in the cerebellum." RNA 8, no. 12 (December 2002): 1538–47. http://dx.doi.org/10.1017/s1355838202028042.
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We compared the gene expression patterns of fetal and adult porcine brains and identified a sequence tag that was more abundant in adult than in fetal brain. The RNA corresponding to the sequence tag has the highest expression level in adult cerebellum. Lower expression levels of the transcript were found in adult cerebrum, pituitary, and uterus, as well as in fetal brain, heart, intestine, kidney, and liver. The sequence tag was used to screen a cDNA library from adult porcine brain. Two independent clones of 2,273 nt and 1,701 nt were isolated. The shorter cDNA is a 5′-truncated form of the longer clone, and both clones have almost identical sequences with multiple start and stop codons in all three reading frames. Screening of two different human brain cDNA libraries with porcine cDNA probes resulted in four overlapping cDNA fragments, which were assembled to one contig of 2,336 nt in length. Like noncoding RNAs, the porcine and human sequences have no common conserved open reading frame and share stretches of high homology interrupted by stretches with almost no homology. The human and porcine RNAs were named UM 9(5)h and UM 9(5)p, respectively. They are part of larger transcripts, which are transcribed from single-copy genes, they have very similar tissue distributions, and their sequences are colinear with the respective genomic fragment.
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KIKONYOGO, Alexandra, and Regina PIETRUSZKO. "Aldehyde dehydrogenase from adult human brain that dehydrogenates γ-aminobutyraldehyde: purification, characterization, cloning and distribution." Biochemical Journal 316, no. 1 (May 15, 1996): 317–24. http://dx.doi.org/10.1042/bj3160317.
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Enzyme purification and characterization, cDNA cloning and Northern blot analysis were the techniques utilized during this investigation to determine the identity and occurrence of the aldehyde dehydrogenase that metabolizes γ-aminobutyraldehyde in adult human brain. The purification yielded one major protein which was active with γ-aminobutyraldehyde. It had the physicochemical and kinetic properties of the human liver E3 isoenzyme of aldehyde dehydrogenase (EC 1.2.1.3), and also interacted with an anti-(liver E3 isoenzyme) antibody. Tryptic peptides derived from the purified brain protein matched the amino acid sequence of the liver E3 isoenzyme. Employing liver E3 cDNA, a human cerebellar cDNA library was screened and a 2.0 kb cDNA fragment was isolated. The cerebellar cDNA yielded a derived primary structure which differed from the liver E3 amino acid sequence by a single serine-to-cysteine substitution at position 88 (position 84 in the liver sequence). Thus the γ-aminobutyraldehyde-metabolizing enzyme from human brain can be identified as E3′, a variant of the E3 isoenzyme. The catalytic properties of the brain variant were indistinguishable from those of E3, and so the functional importance of this variant is at present unknown. The distribution of this enzyme in brain was investigated by Northern blot analysis, which demonstrated the presence of E3′ mRNA in all regions of the human brain. mRNA levels were variable in the different brain areas, with the highest levels in the spinal cord and the lowest in the occipital pole.
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Thomson, S. A. M., E. Kennerly, N. Olby, J. R. Mickelson, D. E. Hoffmann, P. J. Dickinson, G. Gibson, and M. Breen. "Microarray Analysis of Differentially Expressed Genes of Primary Tumors in the Canine Central Nervous System." Veterinary Pathology 42, no. 5 (September 2005): 550–58. http://dx.doi.org/10.1354/vp.42-5-550.
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The pathophysiologic similarities of many human and canine cancers support the role of the domestic dog as a model for brain tumor research. Here we report the construction of a custom canine brain-specific cDNA microarray and the analysis of gene expression patterns of several different types of canine brain tumor The microarray contained 4000 clones from a canine brain specific cDNA library including 2161 clones that matched known genes or expressed sequence tags (ESTs) and 25 cancer-related genes. Our study included 16 brain tumors (seven meningiomas, five glial tumors, two ependymomas, and two choroid plexus papillomas) from a variety of different dog breeds. We identified several genes previously found to be differentially expressed in human brain tumors. This suggests that human and canine brain tumors share a common pathogenesis. In addition, we also found differentially expressed genes unique to either meningiomas or the glial tumors. This report represents the first global gene expression analysis of different types of canine brain tumors by cDNA microarrays and might aid in the identification of potential candidate genes involved in tumor formation and progression.
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Middlemas, D. S., R. A. Lindberg, and T. Hunter. "trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors." Molecular and Cellular Biology 11, no. 1 (January 1991): 143–53. http://dx.doi.org/10.1128/mcb.11.1.143-153.1991.
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We have screened an adult rat cerebellar cDNA library in search of novel protein tyrosine-kinase (PTK) cDNAs. A cDNA for a putative PTK, trkB, was cloned, and its sequence indicates that it is likely to be derived from a gene for a ligand-regulated receptor closely related to the human trk oncogene. Northern (RNA) analysis showed that the trkB gene is expressed predominantly in the brain and that trkB expresses multiple mRNAs, ranging from 0.7 to 9 kb. Hybridization of cerebral mRNAs with a variety of probes indicates that there are mRNAs encoding truncated trkB receptors. Two additional types of cDNA were isolated, and their sequences are predicted to encode two distinct C-terminally truncated receptors which have the complete extracellular region and transmembrane domain, but which differ in their short cytoplasmic tails.
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Middlemas, D. S., R. A. Lindberg, and T. Hunter. "trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors." Molecular and Cellular Biology 11, no. 1 (January 1991): 143–53. http://dx.doi.org/10.1128/mcb.11.1.143.
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We have screened an adult rat cerebellar cDNA library in search of novel protein tyrosine-kinase (PTK) cDNAs. A cDNA for a putative PTK, trkB, was cloned, and its sequence indicates that it is likely to be derived from a gene for a ligand-regulated receptor closely related to the human trk oncogene. Northern (RNA) analysis showed that the trkB gene is expressed predominantly in the brain and that trkB expresses multiple mRNAs, ranging from 0.7 to 9 kb. Hybridization of cerebral mRNAs with a variety of probes indicates that there are mRNAs encoding truncated trkB receptors. Two additional types of cDNA were isolated, and their sequences are predicted to encode two distinct C-terminally truncated receptors which have the complete extracellular region and transmembrane domain, but which differ in their short cytoplasmic tails.
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Gérard, C. M., C. Mollereau, G. Vassart, and M. Parmentier. "Molecular cloning of a human cannabinoid receptor which is also expressed in testis." Biochemical Journal 279, no. 1 (October 1, 1991): 129–34. http://dx.doi.org/10.1042/bj2790129.
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A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).
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Brant, S. R., C. H. Yun, M. Donowitz, and C. M. Tse. "Cloning, tissue distribution, and functional analysis of the human Na+/N+ exchanger isoform, NHE3." American Journal of Physiology-Cell Physiology 269, no. 1 (July 1, 1995): C198—C206. http://dx.doi.org/10.1152/ajpcell.1995.269.1.c198.
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We previously isolated a 1.4-kb partial cDNA from a human kidney cortex library. Using both library screening and reverse transcription-polymerase chain reaction of human kidney RNA, we obtained the entire coding region of the human NHE3 cDNA. The human NHE3 cDNA encoded a protein of 834 amino acids with a calculated relative molecular weight of 92,906. It exhibited 89 and 88% amino acid identity with rat and rabbit NHE3, respectively. The stable transfection of a composite human NHE3 cDNA into Na+/H+ exchanger-deficient PS120 cells established Na+/H+ exchange. Functionally, human NHE3 was similar to the rabbit and rat NHE3 homologues, being relatively resistant to inhibition by amiloride, half-maximal inhibition (IC50) = 49.0 microM, and ethylisopropylamiloride, IC50 = 6.6 microM, and being stimulated by fibroblast growth factor but inhibited by phorbol 12-myristate 13-acetate. However, unlike the rabbit or rat NHE3, human NHE3 message was not restricted to kidney, intestine, stomach, and brain. Northern analysis of multiple human tissues detected NHE3 message, in descending order, as follows: kidney >> small intestine >> testes > ovary > colon = prostate > thymus > peripheral leukocyte = brain > spleen > placenta. Message in the kidney, small intestine, and colon was primarily of 6.7 kb, whereas both 6.7- and 8.9-kb bands were expressed nearly equivalently in the other tissues. No NHE3 message was detected in the human heart, lung, liver, skeletal muscle, or pancreas.
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May, Patrick C., Steven A. Johnson, Judes Poirier, Martha Lampert-Etchells, and Caleb E. Finch. "Altered Gene Expression in Alzheimer's Disease Brain Tissue." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 16, S4 (November 1989): 473–76. http://dx.doi.org/10.1017/s0317167100029796.
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ABSTRACT:We review the evidence for altered gene expression in Alzheimer's disease brain and identify alternative molecular approaches for isolating additional novel markers. One marker, pADHC-9, was isolated from a human hippocampal cDNA library by differential screening with AD and control cDNA probes. This clone hybridizes to a 2 Kb RNA which is increased 2 fold in AD hippocampus. The deduced amino acid sequence of pADHC-9 codes for a 52 kDAL protein similar to a testicular sulfated glycoprotein secreted by rat Sertoli cells. The normal function of this protein in brain and whether that function is altered in Alzheimer's disease is unknown.
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Lewis, S. A., A. Villasante, P. Sherline, and N. J. Cowan. "Brain-specific expression of MAP2 detected using a cloned cDNA probe." Journal of Cell Biology 102, no. 6 (June 1, 1986): 2098–105. http://dx.doi.org/10.1083/jcb.102.6.2098.
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We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.
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BOARD, G. Philip. "Identification of cDNAs encoding two human Alpha class glutathione transferases (GSTA3 and GSTA4) and the heterologous expression of GSTA4-4." Biochemical Journal 330, no. 2 (March 1, 1998): 827–31. http://dx.doi.org/10.1042/bj3300827.
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The Expressed Sequence Tag database has been searched for examples of previously undescribed human Alpha class glutathione transferases. An incomplete transcript of the previously described GSTA3 gene was identified in a cDNA library derived from 8-9 week placenta. This indicates that the GSTA3 gene is functional and is possibly under specific developmental regulation. A second cDNA, termed GSTA4, was identified in a brain cDNA library. The encoded GSTA4-4 enzyme was expressed in Escherichia coli and was found to be immunologically distinct from GSTA1-1 and to have high activity with alk-2-enals. Although GSTA4-4 appears to be functionally similar to the mouse GST5.7 and rat GST8-8 Alpha class enzymes, sequence comparisons and phylogenetic analysis suggest that GSTA4-4 may be a member of a distinct Alpha class subgroup.
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Boado, Ruben J., Jian Yi Li, and William M. Pardridge. "Selective Lutheran Glycoprotein Gene Expression at the Blood—Brain Barrier in Normal Brain and in Human Brain Tumors." Journal of Cerebral Blood Flow & Metabolism 20, no. 7 (July 2000): 1096–102. http://dx.doi.org/10.1097/00004647-200007000-00009.
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The Lutheran (LU) glycoprotein was shown to be a specific marker of brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo. A 1.5 kb partial cDNA encoding the bovine LU was isolated from a bovine brain capillary cDNA library. Sequence analysis showed that the bovine and human LU had a 75% and 79% identity in the amino acid and nucleotide sequences, respectively. Northern blot analysis demonstrated a very high level of gene expression of the LU transcript in freshly isolated bovine brain capillaries, but no measurable LU mRNA in whole bovine brain. The high level of LU gene expression was maintained when bovine brain capillary endothelium was grown in tissue culture. Because many BBB specific genes are downregulated in tissue culture and in brain tumors, the expression of the LU mRNA and immunoactive LU protein was investigated in primary and metastatic human brain tumors. Immunocytochemistry of fresh frozen human brain and human brain tumors showed abundant immunostaining of brain capillary endothelium. Northern blot analysis showed the presence of LU transcripts in a panel of primary and metastatic human brain tumors. These studies demonstrated that the LU glycoprotein was a novel new marker of the BBB, and unlike other BBB specific genes, there was a persistent gene expression of the LU glycoprotein both in brain capillary endothelial cells grown in culture and in the endothelium of capillaries perfusing human brain cancer.
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Johnson, J. L., T. G. Beito, C. J. Krco, and D. O. Toft. "Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes." Molecular and Cellular Biology 14, no. 3 (March 1994): 1956–63. http://dx.doi.org/10.1128/mcb.14.3.1956-1963.1994.
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Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.
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Johnson, J. L., T. G. Beito, C. J. Krco, and D. O. Toft. "Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes." Molecular and Cellular Biology 14, no. 3 (March 1994): 1956–63. http://dx.doi.org/10.1128/mcb.14.3.1956.
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Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.
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Roman, Richard, Andrew P. Feranchak, Marlyn Troetsch, Jeffrey C. Dunkelberg, Gordon Kilic, Thorsten Schlenker, Jerome Schaack, and J. Gregory Fitz. "Molecular characterization of volume-sensitive SKCachannels in human liver cell lines." American Journal of Physiology-Gastrointestinal and Liver Physiology 282, no. 1 (January 1, 2002): G116—G122. http://dx.doi.org/10.1152/ajpgi.2002.282.1.g116.
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In human liver, Ca2+-dependent changes in membrane K+permeability play a central role in coordinating functional interactions between membrane transport, metabolism, and cell volume. On the basis of the observation that K+conductance is partially sensitive to the bee venom toxin apamin, we aimed to assess whether small-conductance Ca2+-sensitive K+(SKCa) channels are expressed endogenously and contribute to volume-sensitive K+efflux and cell volume regulation. We isolated a full-length 2,140-bp cDNA (hSK2) highly homologous to rat brain rSK2 cDNA, including the putative apamin-sensitive pore domain, from a human liver cDNA library. Identical cDNAs were isolated from primary human hepatocytes, human HuH-7 hepatoma cells, and human Mz-ChA-1 cholangiocarcinoma cells. Transduction of Chinese hamster ovary cells with a recombinant adenovirus encoding the hSK2-green fluorescent protein fusion construct resulted in expression of functional apamin-sensitive K+channels. In Mz-ChA-1 cells, hypotonic (15% less sodium glutamate) exposure increased K+current density (1.9 ± 0.2 to 37.5 ± 7.1 pA/pF; P < 0.001). Apamin (10–100 nM) inhibited K+current activation and cell volume recovery from swelling. Apamin-sensitive SKCachannels are functionally expressed in liver and biliary epithelia and likely contribute to volume-sensitive changes in membrane K+permeability. Accordingly, the hSK2 protein is a potential target for pharmacological modulation of liver transport and metabolism through effects on membrane K+permeability.
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GÓMEZ-FABRE, Pedro M., Juan C. ALEDO, Antonio DEL CASTILLO-OLIVARES, Francisco J. ALONSO, Ignacio NÚÑEZ DE CASTRO, José A. CAMPOS, and Javier MÁRQUEZ. "Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase." Biochemical Journal 345, no. 2 (January 10, 2000): 365–75. http://dx.doi.org/10.1042/bj3450365.
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Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of λgt10 cDNA library screening and the rapid amplification of cDNA ends (‘RACE’) technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.
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Sasaki, Y. F. "Construction of an Equalized cDNA Library from Human Brain by Semi-Solid Self-Hybridization System." DNA Research 1, no. 2 (January 1, 1994): 91–96. http://dx.doi.org/10.1093/dnares/1.2.91.
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Chu, Chun, Jian Yi Li, Ruben J. Boado, and William M. Pardridge. "Blood—Brain Barrier Genomics and Cloning of a Novel Organic Anion Transporter." Journal of Cerebral Blood Flow & Metabolism 28, no. 2 (August 1, 2007): 291–301. http://dx.doi.org/10.1038/sj.jcbfm.9600538.
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A novel organic anion transporter selectively expressed at the blood—brain barrier (BBB), originally designated BBB-specific anion transporter type 1 (BSAT1), and now classified as Slco1c1, has been cloned from a BBB genomics program as a partial cDNA; this study describes the cloning and expression of the full-length cDNA from a rat brain capillary cDNA library. Northern analysis revealed the selective expression of the transporter at the BBB, and the transporter was expressed after permanent transfection of human 293 cells with cDNA encoding either the full length or open reading frame mRNA. The full-length transporter cDNA was 2.6 kb, and the mRNA was highly expressed at the rat brain microvasculature, but not in kidney, liver, heart, or lung, or in glial cells or brain glial tumors. Blood—brain barrier-specific anion transporter type 1 expression in 293 cells was poor after the transfection of the full-length cDNA, whereas transporter expression in 293 cells was high after transfection of the open reading frame. The transporter showed asymmetric kinetic properties in comparison of the influx and efflux of model substrates, thyroxine (T4), triiodothyronine (T3), and estradiol-glucuronide (E2G). Thyroxine and T3 inhibited the influx of E2G, but E2G did not inhibit thyroxine influx, and T3 only weakly inhibited the influx of T4. Extracellular E2G stimulated the transefflux of intracellular T4. Blood—brain barrier-specific anion transporter type 1 is a novel organic anion transporter that is a sodium-independent exchanger that may participate in the active efflux of iodothyronines and steroid conjugates at the BBB.
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Lewis, S. A., P. Sherline, and N. J. Cowan. "A cloned cDNA encoding MAP1 detects a single copy gene in mouse and a brain-abundant RNA whose level decreases during development." Journal of Cell Biology 102, no. 6 (June 1, 1986): 2106–14. http://dx.doi.org/10.1083/jcb.102.6.2106.
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Screening of a bacteriophage lambda gt11 cDNA expression library with a polyclonal anti-microtubule associated protein (MAP) antiserum resulted in the isolation of two non-cross-hybridizing sets of cDNA clones. One set was shown to encode MAP2 (Lewis, S. A., A. Villasante, P. Sherline, and N. J. Cowan, 1986, J. Cell Biol., 102:2098-2105). To determine the specificity of the second set, three non-overlapping fragments cloned from the same mRNA molecule via a series of "walking" experiments were separately subcloned into inducible plasmid expression vectors in the appropriate orientation and reading frame. Upon induction and analysis by immunoblotting, two of the fusion proteins synthesized were shown to be immunoreactive with an anti-MAP1-specific antibody, but not with an anti-MAP2-specific antibody. Since these MAP1-specific epitopes are encoded in non-overlapping cDNAs cloned from a single contiguous mRNA, these clones cannot encode polypeptides that contain adventitiously cross-reactive epitopes. Furthermore, these cDNA clones detected an abundant mRNA species of greater than 10 kb in mouse brain, consistent with the coding requirement of a 350,000-D polypeptide and the known abundance of MAP1 in that tissue. The MAP1-specific cDNA probes were used in blot transfer experiments with RNA prepared from brain, liver, kidney, stomach, spleen, and thymus. While detectable quantities of MAP1-specific mRNA were observed in these tissues, the level of MAP1 expression was approximately 500-fold lower than in brain. The levels of both MAP1-specific and MAP2-specific mRNAs decline in the postnatal developing brain; the level of MAP1-specific mRNA also increases slightly in rat PC12 cells upon exposure to nerve growth factor. These surprising results contrast sharply with reported dramatic developmental increases in the amount of MAP1 in brain and in nerve growth factor-induced PC12 cells. The cDNA clones encoding MAP1 detect a single copy sequence in mouse DNA, even under conditions of low stringency that would allow the detection of related but mismatched sequences. The cDNAs cross-hybridize with genomic sequences in rat, human, and chicken DNA, but not with DNA from frog, Drosophila, or sea urchin. These data are discussed in terms of the evolution and possible biological role of MAP1.
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Pelletier, Glenn J., Steven L. Brody, Helen Liapis, Robert A. White, and Brian P. Hackett. "A human forkhead/winged-helix transcription factor expressed in developing pulmonary and renal epithelium." American Journal of Physiology-Lung Cellular and Molecular Physiology 274, no. 3 (March 1, 1998): L351—L359. http://dx.doi.org/10.1152/ajplung.1998.274.3.l351.
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Members of the forkhead/winged-helix transcription factor family play crucial roles during vertebrate development. A human hepatocyte nuclear factor/forkhead homolog (HFH)-4 cDNA encoding a 421-amino acid protein was isolated from a human fetal lung cDNA library. By Southern blot analysis of human-rodent somatic cell hybrid genomic DNA, the human HFH-4 gene localizes to chromosome 17q23-qter. This is the locus of another forkhead/winged-helix gene, the interleukin enhancer binding factor gene. RNA blot analysis revealed a 2.5-kilobase human HFH-4 transcript in fetal lung, kidney, and brain as well as in adult reproductive tissues, lung, and brain. By in situ hybridization, HFH-4 expression is associated with differentiation of the proximal pulmonary epithelium, starting during the pseudoglandular stage of human lung development. During human renal morphogenesis, HFH-4 is expressed in the developing epithelial cells of the ureteric duct, glomerulus, and epithelial vesicles. The unique pattern of HFH-4 expression during human fetal development suggests a role for this forkhead/winged-helix factor during pulmonary and renal epithelial development.
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Cheetham, M. E., J. P. Brion, and B. H. Anderton. "Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons." Biochemical Journal 284, no. 2 (June 1, 1992): 469–76. http://dx.doi.org/10.1042/bj2840469.
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The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation. The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK. Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described. We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues. The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease. The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70. Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal. These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms.
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Lewis, S. A., and N. J. Cowan. "Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe." Journal of Cell Biology 100, no. 3 (March 1, 1985): 843–50. http://dx.doi.org/10.1083/jcb.100.3.843.
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A 1.2-kilobase (kb) cDNA clone (NF68) encoding the mouse 68,000-mol-wt neurofilament protein is described. The clone was isolated from a mouse brain cDNA library by low-stringency cross-hybridization with a cDNA probe encoding mouse glial fibrillary acidic protein (Lewis et al., 1984, Proc. Natl. Acad. Sci. USA., 81:2743-2746). The identity of NF68 was established by hybrid selection using mouse brain polyA+ mRNA, and cell-free translation of the selected mRNA species. The cell-free translation product co-migrated with authentic 68,000-mol-wt neurofilament protein on an SDS/polyacrylamide gel, and was immunoprecipitable with a monospecific rabbit anti-bovine neurofilament antiserum. In addition, DNA sequence analysis of NF68 showed 90% homology at the amino acid level compared with the sequence of the porcine 68,000-mol-wt neurofilament protein. At high stringency, NF68 detects a single genomic sequence encoding the mouse 68,000-mol-wt neurofilament protein. Two mRNA species of 2.5 kb and 4.0 kb are transcribed from the single gene in mouse brain. The level of expression of these mRNAs remains almost constant in postnatal mouse brains of all ages and, indeed, in the adult. At reduced stringency, NF68 detects a number of mRNAs that are expressed in mouse brain, one of which encodes the 150,000-mol-wt neurofilament protein. The NF68 probe cross-hybridizes at high stringency with genomic sequences in species as diverse as human, chicken, and (weakly) frog, but not with DNA from Drosophila or sea urchin.
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BECCARI, T., M. G. APPOLLONI, E. COSTANZI, S. STINCHI, J. L. STIRLING, M. A. DELLA FAZIA, G. SERVILLO, M. P. VIOLA, and A. ORLACCHIO. "Lysosomal α-mannosidases of mouse tissues: characteristics of the isoenzymes, and cloning and expression of a full-length cDNA." Biochemical Journal 327, no. 1 (October 1, 1997): 45–49. http://dx.doi.org/10.1042/bj3270045.
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Lysosomal α-D-mannosidase from mouse tissues was separated into its constituent isoenzymes by DEAE-cellulose chromatography. Forms corresponding to the human isoenzymes B and A were present in testis, brain, spleen and kidney, whereas in epididymis and liver only the B form was present. Murine α-mannosidases A and B are glycoproteins and have pH optima, thermal stabilities and molecular masses similar to those of the human isoenzymes. A full-length cDNA (3.1 kb) containing the complete coding sequence for α-mannosidase was isolated from a mouse macrophage cDNA library. Comparison of the deduced amino acid sequences of human and mouse α-mannosidases showed that they had 75% identity and 83% similarity. Expression of this cDNA in COS cells showed that both the A and the B isoenzymes can arise from a single transcript. Northern blotting analysis showed a 10-fold range in the abundance of α-mannosidase mRNA in mouse tissues, with the highest levels found in epididymis, and the lowest in liver.
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Ramamoorthy, S., F. H. Leibach, V. B. Mahesh, H. Han, T. Yang-Feng, R. D. Blakely, and V. Ganapathy. "Functional characterization and chromosomal localization of a cloned taurine transporter from human placenta." Biochemical Journal 300, no. 3 (June 15, 1994): 893–900. http://dx.doi.org/10.1042/bj3000893.
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A cDNA clone highly related to the rat brain taurine transporter has been isolated from a human placental cDNA library. Transfection of this cDNA into HeLa cells results in a marked elevation of taurine transport activity. The activity of the cDNA-induced transporter is dependent on the presence of Na+ as well as Cl-. The Na+/Cl-/taurine stoichiometry for the cloned transporter is 2:1:1. The transporter is specific for taurine and other beta-amino acids, including beta-alanine, and exhibits high affinity for taurine (Michaelis-Menten constant approximately 6 microM). The clone consists of a coding region 1863 bp long (including the termination codon), flanked by a 376 bp-long 5′ non-coding region and a 625 bp-long 3′ non-coding region. The nucleotide sequence of the coding region predicts a 620-amino acid protein with a calculated M(r) of 69,853. Northern-blot analysis of poly(A)+ RNA from several human tissues indicates a complex expression pattern differing across tissues. The principal transcript, 6.9 kb in size, is expressed abundantly in placenta and skeletal muscle, at intermediate levels in heart, brain, lung, kidney and pancreas and at low levels in liver. Cultured human cell lines derived from placenta (JAR and BeWo), intestine (HT-29), cervix (HeLa) and retinal pigment epithelium (HRPE), which are known to possess Na(+)- and Cl(-)-coupled taurine transport activity, also contain the 6.9 kb transcript. Somatic cell hybrid and in situ hybridization studies indicate that the cloned taurine transporter is localized to human chromosome 3 p24-->p26.
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Yi, Joo‐Mi, Kyung‐Mi Shin, Ji‐Won Lee, In‐Ho Paik, Kyung‐Lib Jang, and Heui‐Soo Kim. "Identification and phylogenetic analysis of sine‐R Retroposon family in cDNA Library of human fetal brain." Korean Journal of Biological Sciences 5, no. 3 (January 2001): 231–36. http://dx.doi.org/10.1080/12265071.2001.9647608.
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Hosoyamada, Makoto, Takashi Sekine, Yoshikatsu Kanai, and Hitoshi Endou. "Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney." American Journal of Physiology-Renal Physiology 276, no. 1 (January 1, 1999): F122—F128. http://dx.doi.org/10.1152/ajprenal.1999.276.1.f122.
Full textAbstract:
Recently, we isolated the multispecific organic anion transporter (OAT1) from the rat kidney, which plays important roles in the renal elimination of endogenous and exogenous organic anions including clinically important drugs. In the present study, we cloned and characterized human OAT1. Two cDNA clones, hOAT1–1 cDNA and hOAT1–2 cDNA, were isolated from a human kidney cDNA library, whose amino acid sequences were 86.0% and 87.8% identical to that of rat OAT1, respectively. When expressed in Xenopus laevis oocytes, hOAT1 mediated sodium-independent uptake of p-aminohippurate (PAH) ( K m = 9.3 ± 1.0 μM). hOAT1-mediated PAH uptake was inhibited by bulky inorganic anions, various xenobiotics, and endogenous substances, including benzylpenicillin, furosemide, indomethacin, probenecid, phenol red, urate, and α-ketoglutarate. Northern blot analysis revealed that hOAT1 mRNA is strongly expressed in human kidney; transcripts of different sizes are expressed in skeletal muscle, brain, and placenta. Immunohistochemical analysis using rabbit IgG antibody against the carboxy-terminal 14 peptides of hOAT1 revealed that hOAT1 is expressed at the basolateral membrane of the proximal tubule. hOAT1 gene was located on human chromosome 11q13.1 by fluorescent in situ hybridization analysis. These results indicate that hOAT1 is a multispecific organic anion transporter on the basolateral membrane of the proximal tubule in human kidney.
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Leto, T. L., D. Fortugno-Erikson, D. Barton, T. L. Yang-Feng, U. Francke, A. S. Harris, J. S. Morrow, V. T. Marchesi, and E. J. Benz. "Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species." Molecular and Cellular Biology 8, no. 1 (January 1988): 1–9. http://dx.doi.org/10.1128/mcb.8.1.1-9.1988.
Full textAbstract:
The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.
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Leto, T. L., D. Fortugno-Erikson, D. Barton, T. L. Yang-Feng, U. Francke, A. S. Harris, J. S. Morrow, V. T. Marchesi, and E. J. Benz. "Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species." Molecular and Cellular Biology 8, no. 1 (January 1988): 1–9. http://dx.doi.org/10.1128/mcb.8.1.1.
Full textAbstract:
The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.
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Meléndez, Jorge, Vilma Maldonado, Collin D. Bingle, Moisés Selman, and Annie Pardo. "Cloning and expression of guinea pig TIMP-2. Expression in normal and hyperoxic lung injury." American Journal of Physiology-Lung Cellular and Molecular Physiology 278, no. 4 (April 1, 2000): L737—L743. http://dx.doi.org/10.1152/ajplung.2000.278.4.l737.
Full textAbstract:
Tissue inhibitors of metalloproteinases (TIMPs) play a key regulatory role in extracellular matrix remodeling. By screening a lung library with a human TIMP-2 cDNA probe, we have isolated the cDNA corresponding to guinea pig TIMP-2. The 3.5-kb cDNA presents an open reading frame that predicts a protein of 220 amino acids showing 97.2, 96.8, 97.2, and 77.3% overall identity with human, mouse, rat, and chicken TIMP-2, respectively. Guinea pig TIMP-2 cDNA was expressed in CHO-K1 cells, showing a protein with the expected molecular weight and activity. Northern blot analysis revealed TIMP-2 expression in brain, kidney, intestine, spleen, heart, and lung. Transforming growth factor-β downregulated TIMP-2 mRNA in guinea pig lung fibroblasts, whereas a variety of other stimuli showed no effect. In normal and hyperoxia-exposed lungs, TIMP-2 mRNA was mainly localized in alveolar macrophages and epithelial cells. No quantitative differences were found by Northern blot. These results confirm that TIMP-2 is highly conserved in mammals and largely expressed in lungs.
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Chu, FF, RS Esworthy, JH Doroshow, K. Doan, and XF Liu. "Expression of plasma glutathione peroxidase in human liver in addition to kidney, heart, lung, and breast in humans and rodents." Blood 79, no. 12 (June 15, 1992): 3233–38. http://dx.doi.org/10.1182/blood.v79.12.3233.3233.
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Abstract We analyzed the expression of plasma glutathione peroxidase (GSHPx-P) messenger RNA (mRNA) in mouse, rat, and human tissues, using a human GSHPx-P cDNA clone as the probe. Unlike the classical cellular glutathione peroxidase (GSHPx-1), GSHPx-P expression appears to be tissue-specific. In the mouse and rat, kidney expresses an mRNA at a high level detected with the human probe. A signal is also detected in mRNA isolated from mouse and rat heart, rat cardiac myocytes, mouse lung, epididymis, and the mammary gland of midpregnant mice. No signal is detected in mRNA isolated from mouse and rat liver, mouse brain, uterus, and testis. In human tissues, an mRNA hybridizing to GSHPx-P cDNA is present in liver, as well as kidney, heart, lung, breast, and placenta. We have shown that human kidney expresses a GSHPx-P mRNA, and not a GSHPx-P-like message, by isolating a cDNA clone from a human kidney library in lambda gt11. From the 412-nucleotide partial sequence of the kidney cDNA, which codes for the 40–170 amino acids of GSHPx-P including the TGA codon for selenocysteine, we found complete sequence identity of the kidney cDNA with GSHPx-P isolated from placenta. The expression of GSHPx-P mRNA in cell lines was also studied. There is some correlation of the expression of GSHPx-P in these cell lines with that in normal tissues. Cell lines that expressed GSHPx-P mRNA or protein included the human hepatocarcinoma HepG2, Hep3B cells, human kidney carcinoma A498 cells, and the human breast cancer SK-BR-3, T47D, MDA-MB-231, and AdrrMCF-7 cells. Cell lines that did not express GSHPx- P included human choriocarcinoma BeWo cells, human breast cancer MCF-7, ZR-75–1, and Hs578T cells, and mouse hepatoma Hepa-1 cells.
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Chu, FF, RS Esworthy, JH Doroshow, K. Doan, and XF Liu. "Expression of plasma glutathione peroxidase in human liver in addition to kidney, heart, lung, and breast in humans and rodents." Blood 79, no. 12 (June 15, 1992): 3233–38. http://dx.doi.org/10.1182/blood.v79.12.3233.bloodjournal79123233.
Full textAbstract:
We analyzed the expression of plasma glutathione peroxidase (GSHPx-P) messenger RNA (mRNA) in mouse, rat, and human tissues, using a human GSHPx-P cDNA clone as the probe. Unlike the classical cellular glutathione peroxidase (GSHPx-1), GSHPx-P expression appears to be tissue-specific. In the mouse and rat, kidney expresses an mRNA at a high level detected with the human probe. A signal is also detected in mRNA isolated from mouse and rat heart, rat cardiac myocytes, mouse lung, epididymis, and the mammary gland of midpregnant mice. No signal is detected in mRNA isolated from mouse and rat liver, mouse brain, uterus, and testis. In human tissues, an mRNA hybridizing to GSHPx-P cDNA is present in liver, as well as kidney, heart, lung, breast, and placenta. We have shown that human kidney expresses a GSHPx-P mRNA, and not a GSHPx-P-like message, by isolating a cDNA clone from a human kidney library in lambda gt11. From the 412-nucleotide partial sequence of the kidney cDNA, which codes for the 40–170 amino acids of GSHPx-P including the TGA codon for selenocysteine, we found complete sequence identity of the kidney cDNA with GSHPx-P isolated from placenta. The expression of GSHPx-P mRNA in cell lines was also studied. There is some correlation of the expression of GSHPx-P in these cell lines with that in normal tissues. Cell lines that expressed GSHPx-P mRNA or protein included the human hepatocarcinoma HepG2, Hep3B cells, human kidney carcinoma A498 cells, and the human breast cancer SK-BR-3, T47D, MDA-MB-231, and AdrrMCF-7 cells. Cell lines that did not express GSHPx- P included human choriocarcinoma BeWo cells, human breast cancer MCF-7, ZR-75–1, and Hs578T cells, and mouse hepatoma Hepa-1 cells.
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Piétu, Geneviève, Régine Mariage-Samson, Nicole-Adeline Fayein, Christiane Matingou, Eric Eveno, Rémi Houlgatte, Charles Decraene, et al. "The Genexpress IMAGE Knowledge Base of the Human Brain Transcriptome: A Prototype Integrated Resource for Functional and Computational Genomics." Genome Research 9, no. 2 (February 1, 1999): 195–209. http://dx.doi.org/10.1101/gr.9.2.195.
Full textAbstract:
Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the addresshttp://idefix.upr420.vjf.cnrs.fr/EXPR/welcome.html.
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WALKLEY, Neal A., Andrew G. DEMAINE, and Afshan N. MALIK. "Cloning, structure and mRNA expression of human Cctg, which encodes the chaperonin subunit CCTγ." Biochemical Journal 313, no. 2 (January 15, 1996): 381–89. http://dx.doi.org/10.1042/bj3130381.
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We describe the cloning, DNA sequence analysis and mRNA expression analysis of human Cctg (HsCctg), a gene that encodes the γ-subunit of the eukaryotic cytosolic ‘chaperonin-containing TCP-1’ (CCT). Partial clones representing the 3ʹ region of HsCctg cDNA were isolated from a human kidney cDNA library, and the missing 5ʹ region was amplified directly from human kidney cDNA. The Cctg mRNA transcript is expressed in numerous human and mouse tissues and, like Tcp-1/Ccta, Cctg mRNA is expressed at higher levels in mouse testis when compared with kidney and brain. Southern-blot analysis has also revealed the Cctg gene to be highly conserved in mouse, rat, sheep and frog. The 1901 bp HsCctg cDNA has a coding region of 1635 bp and encodes a predicted 60 kDa protein (544 amino acids). The predicted HsCCTγ amino acid sequence shares a high degree of sequence similarity with γ-subunits from the mouse Mus musculus (98% similarity), the yeast Saccharomyces cerevisiae (75% similarity) and the protozoan Tetrahymena pyriformis (76% similarity) as well as with other members of the TF55/TCP-1 family, such as human TCP-1/CCTα (55% similarity) and TCP-20/CCTζ (54% similarity). HsCCTγ also shares conserved domains previously identified in the TF55/TCP-1 family of chaperonins and more distantly related chaperonins such as GroEL and Hsp60.
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Munguia, Maria Elena, Tzipe Govezensky, Rodrigo Martinez, Karen Manoutcharian, and Goar Gevorkian. "Identification of amyloid-beta 1–42 binding protein fragments by screening of a human brain cDNA library." Neuroscience Letters 397, no. 1-2 (April 2006): 79–82. http://dx.doi.org/10.1016/j.neulet.2005.11.061.
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Himmler, A., D. Drechsel, M. W. Kirschner, and D. W. Martin. "Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains." Molecular and Cellular Biology 9, no. 4 (April 1989): 1381–88. http://dx.doi.org/10.1128/mcb.9.4.1381-1388.1989.
Full textAbstract:
Tau proteins consist of a family of proteins, heterogeneous in size, which associate with microtubules in vivo and are induced during neurite outgrowth. In humans, tau is one of the major components of the pathognomonic neurofibrillary tangles in Alzheimer's disease brain. Screening of a cDNA library prepared from bovine brain led to the isolation of several cDNA clones encoding tau proteins with different N termini and differing by insertions or deletions, suggesting differential splicing of the tau transcripts. One of the N-terminal domains and the repeated C-terminal domain of the encoded tau proteins are recognized by polyclonal antibodies to bovine tau. The bovine tau proteins are highly homologous to murine and human tau, especially within the repeated C-terminal domain. Compared with murine and human tau, bovine tau contains the insertion of three longer segments, one of which is an additional characteristic repeat. Portions of tau proteins generated by in vitro translation were used to show that these repeats represent tubulin-binding domains, two of which are sufficient to bind to microtubules assembled from purified tubulin in the presence of taxol.
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Himmler, A., D. Drechsel, M. W. Kirschner, and D. W. Martin. "Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains." Molecular and Cellular Biology 9, no. 4 (April 1989): 1381–88. http://dx.doi.org/10.1128/mcb.9.4.1381.
Full textAbstract:
Tau proteins consist of a family of proteins, heterogeneous in size, which associate with microtubules in vivo and are induced during neurite outgrowth. In humans, tau is one of the major components of the pathognomonic neurofibrillary tangles in Alzheimer's disease brain. Screening of a cDNA library prepared from bovine brain led to the isolation of several cDNA clones encoding tau proteins with different N termini and differing by insertions or deletions, suggesting differential splicing of the tau transcripts. One of the N-terminal domains and the repeated C-terminal domain of the encoded tau proteins are recognized by polyclonal antibodies to bovine tau. The bovine tau proteins are highly homologous to murine and human tau, especially within the repeated C-terminal domain. Compared with murine and human tau, bovine tau contains the insertion of three longer segments, one of which is an additional characteristic repeat. Portions of tau proteins generated by in vitro translation were used to show that these repeats represent tubulin-binding domains, two of which are sufficient to bind to microtubules assembled from purified tubulin in the presence of taxol.
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Cioe, L., P. Laurila, P. Meo, K. Krebs, S. Goodman, and PJ Curtis. "Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA." Blood 70, no. 4 (October 1, 1987): 915–20. http://dx.doi.org/10.1182/blood.v70.4.915.915.
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Abstract A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta- spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.
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Cioe, L., P. Laurila, P. Meo, K. Krebs, S. Goodman, and PJ Curtis. "Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA." Blood 70, no. 4 (October 1, 1987): 915–20. http://dx.doi.org/10.1182/blood.v70.4.915.bloodjournal704915.
Full textAbstract:
A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta- spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.
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HOWARD, Linda, Xiaohong LU, Sally MITCHELL, Susan GRIFFITHS, and Paul GLYNN. "Molecular cloning of MADM: a catalytically active mammalian disintegrin-metalloprotease expressed in various cell types." Biochemical Journal 317, no. 1 (July 1, 1996): 45–50. http://dx.doi.org/10.1042/bj3170045.
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A peptide sequence of a metalloprotease purified from bovine brain [Chantry, Gregson and Glynn (1989) J. Biol. Chem. 264, 21603–21607] was used to design an oligonucleotide probe for screening a bovine brain cDNA library. A contig of the two overlapping cDNA clones that were isolated encoded a 748-amino-acid polypeptide with similarity to the disintegrin–metalloprotease precursor proteins of haemorrhagic snake venom. The bovine protein has been named MADM, for mammalian disintegrin–metalloprotease. The predicted mature protein has 534 amino acids arrayed as extracellular metalloprotease and disintegrin (potential integrin-binding) domains, a transmembrane helix and a basic/proline-rich cytoplasmic C-terminus. Highly conserved homologues of bovine MADM were found in cDNA libraries of rat brain and a human U937 histiocytic lymphoma cell line. A wide variety of mammalian cell lines expressed low levels of MADM mRNA (4.5 and 3.2 kb transcripts) and mature polypeptide (Mr 62000), as assessed by Northern analysis and Western blotting with an antiserum raised to a peptide within the disintegrin domain. MADM appears to be a rather distantly related member of the reprolysin protein family, which includes both the snake venom disintegrin–metalloproteases and a number of predicted cell-surface disintegrin-containing mammalian proteins.
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Wojcik, SF, FL Schanbacher, LK McCauley, H. Zhou, V. Kartsogiannis, CC Capen, and TJ Rosol. "Cloning of bovine parathyroid hormone-related protein (PTHrP) cDNA and expression of PTHrP mRNA in the bovine mammary gland." Journal of Molecular Endocrinology 20, no. 2 (April 1, 1998): 271–80. http://dx.doi.org/10.1677/jme.0.0200271.
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Parathyroid hormone-related protein (PTHrP) produced by the mammary gland has been postulated to have multiple functions in both the mother and neonate. In humans, alternative 3'-mRNA splicing and endoproteolytic processing result in multiple bioactive PTHrP peptides. Multiple PTHrP peptides also have been reported in bovine milk. To investigate the source of molecular heterogeneity of PTHrP in bovine milk, bovine PTHrP was cloned from a bovine brain cDNA library, sequenced and used to characterize the mammary PTHrP transcript. A 1065 bp clone (bP1) for bovine PTHrP was isolated from a brain cDNA library. The bP1 clone contained the entire coding sequence of PTHrP and 61 and 473 nucleotides of the 5'- and 3'-untranslated regions (UTRs) respectively. The predicted amino acid sequence of bovine PTHrP was 72-92% homologous to the sequences of chicken, rat, mouse, human, and canine PTHrP with the highest sequence divergence present in the C-terminal region of the peptide. The 5'- and 3'-UTRs of bovine brain PTHrP have a high degree of homology to exons 4 and 9 of human PTHrP respectively. PTHrP was expressed as a single 1200 nucleotide mRNA transcript in lactating bovine mammary tissue. RT-PCR using region-specific oligonucleotide primers derived from bP1 demonstrated that PTHrP mRNA transcripts in bovine brain and lactating mammary gland utilize the same 5'- and 3'-UTRs. Expression of PTHrP mRNA was localized to secretory and ductular epithelial cells within the lactating mammary gland, as detected using in situ hybridization. Expression of PTHrP mRNA was demonstrated in the mammary gland during late pregnancy and throughout lactation in cows.
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Rand, E. B., A. M. Depaoli, N. O. Davidson, G. I. Bell, and C. F. Burant. "Sequence, tissue distribution, and functional characterization of the rat fructose transporter GLUT5." American Journal of Physiology-Gastrointestinal and Liver Physiology 264, no. 6 (June 1, 1993): G1169—G1176. http://dx.doi.org/10.1152/ajpgi.1993.264.6.g1169.
Full textAbstract:
cDNA clones encoding rat GLUT5-small intestinal facilitative hexose transporter were isolated from a jejunum library by cross-hybridization with a human GLUT5 cDNA probe. The cDNA sequence indicates that rat GLUT5 is composed of 502 amino acids and has 81.5% amino acid identity and 87.3% similarity with the sequence of human GLUT5. Expression of synthetic rat GLUT5 mRNA in Xenopus oocytes showed that rat GLUT5 was able to mediate the uptake of fructose and, to a lesser extent, of glucose. RNA blotting studies showed that GLUT5 mRNA was present in rat small intestine, kidney, and brain. Although GLUT5 mRNA is expressed in human testis, adipose tissue, and skeletal muscle, it could not be detected by RNA blotting in these rat tissues. Developmental studies showed low levels of GLUT5 mRNA in rat small intestine and kidney during the prenatal period with a rapid induction of GLUT5 expression occurring postnatally. In situ hybridization studies of GLUT5 mRNA expression in the small intestine revealed differential expression along the crypt-villus axis with the highest levels of mRNA being in the midvillus region. In addition, there was quantitatively more GLUT5 mRNA detected in the proximal as opposed to the distal small intestine.
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Rabin, D. U., S. M. Pleasic, J. A. Shapiro, H. Yoo-Warren, J. Oles, J. M. Hicks, D. E. Goldstein, and P. M. Rae. "Islet cell antigen 512 is a diabetes-specific islet autoantigen related to protein tyrosine phosphatases." Journal of Immunology 152, no. 6 (March 15, 1994): 3183–88. http://dx.doi.org/10.4049/jimmunol.152.6.3183.
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Abstract Islet cell Ag 512 (ICA512) is a recombinant human Ag that was isolated from an islet cDNA expression library by screening with human insulin-dependent diabetes mellitus sera. Specificity of reaction with diabetic sera was demonstrated initially by immunoprecipitation with a small number of diabetic and normal serum samples. To permit quantitative and rapid serum testing, ICA512 was purified and adapted to an ELISA format. In this way, a sensitivity of 48% with newly diagnosed diabetic sera has been measured with a panel of 80 sera. DNA sequencing of ICA512-3, a cDNA that contains a 1644 bp open reading frame, suggests that it codes for a transmembrane protein having a single membrane-spanning segment and a cytoplasmic domain that is closely related to the first intracellular (catalytic) domain of the T cell protein tyrosine phosphatase, CD45. Northern blot analysis of poly(A)+ RNAs from several human tissues indicates that ICA512 mRNA is expressed in brain and pancreas.
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Barton, C. H., G. Dickson, H. J. Gower, L. H. Rowett, W. Putt, V. Elsom, S. E. Moore, C. Goridis, and F. S. Walsh. "Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle." Development 104, no. 1 (September 1, 1988): 165–73. http://dx.doi.org/10.1242/dev.104.1.165.
Full textAbstract:
Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 × 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 × 10(3). The size difference between the 125 × 10(3). The size difference between the 125 × 10(3) Mr skeletal muscle form and the 120 × 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 × 10(3) which is processed by microsomal membranes to yield a 122 × 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 × 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.
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Vanweyenberg, V., D. Communi, C. S. D'Santos, and C. Erneux. "Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes." Biochemical Journal 306, no. 2 (March 1, 1995): 429–35. http://dx.doi.org/10.1042/bj3060429.
Full textAbstract:
The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.
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Li, Xin, Lei Chen, Chaoneng Ji, Bing Liu, Jiefeng Gu, Jian Xu, Xianqiong Zou, Shaohua Gu, and Yumin Mao. "Isolation and expression pattern of RGS21 gene, a novel RGS member." Acta Biochimica Polonica 52, no. 4 (September 8, 2005): 943–46. http://dx.doi.org/10.18388/abp.2005_3412.
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Regulators of G-protein signaling (RGS) proteins are known for the RGS domain that is composed of a conserved stretch of 120 amino acids, which binds directly to activated G-protein alpha subunits and acts as a GTPase-activating protein (GAP), leading to their deactivation and termination of downstream signals. In this study, a novel human RGS cDNA (RGS21), 1795 bp long and encoding a 152-amino acid polypeptide, was isolated by large-scale sequencing analysis of a human fetal brain cDNA library. Unlike other RGS family members, RGS21 gene has no additional domain/motif and may represent the smallest known member of RGS family. It may belong to the B/R4 subfamily, which suggests that it may serve exclusively as a negative regulator of alphai/o family members and/or alphaq/11. PCR analysis showed that RGS21 mRNA was expressed ubiquitously in the 16 tissues examined, implying general physiological roles.
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Schoen, T. J., K. Mazuruk, R. J. Waldbillig, J. Potts, D. C. Beebe, G. J. Chader, and I. R. Rodriguez. "Cloning and characterization of a chick embryo cDNA and gene for IGF-binding protein-2." Journal of Molecular Endocrinology 15, no. 1 (August 1995): 49–59. http://dx.doi.org/10.1677/jme.0.0150049.
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ABSTRACT We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1·6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5′ end, the complete coding sequence was obtained from 5′ rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted Mr of 33 500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2·3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.
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van Hille, B., H. Richener, P. Schmid, I. Puettner, J. R. Green, and G. Bilbe. "Heterogeneity of vacuolar H+-ATPase: differential expression of two human subunit B isoforms." Biochemical Journal 303, no. 1 (October 1, 1994): 191–98. http://dx.doi.org/10.1042/bj3030191.
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The catalytic domain of the vacuolar proton ATPase is composed of a hexamer of three A subunits and three B subunits. Here we describe the cloning and characterization of a cDNA isoform of subunit B, HO57, from an osteoclastoma cDNA library. HO57 is represented by three species of mRNA of 1.6, 2.6 and 2.8 kb and is expressed at low levels in a range of human tissues, but at significantly higher levels in brain, kidney and osteoclastoma, and is probably an ubiquitously expressed isoform. In contrast, the kidney-specific isoform has an mRNA of 2 kb and is specifically expressed at high levels only in kidney and, at a lower level, in placenta. Thus the HO57 isoform is integral to the vacuolar ATPase found in the general secretory system of all cells as well as in vacuolar-ATPase-rich sources such as neurones and osteoclasts, whereas both the kidney-specific isoform and HO57 are highly expressed in the kidney. Furthermore, we show by in situ hybridization that HO57 is the only isoform that is exclusively and highly expressed by osteoclasts.