Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Carp acclimated to 10 degrees C gave 69k, 66k, and 62kDa light meromyosin (LMM) fragments in SDS-PAGE, while fish acclimated to 30 degrees C gave 74k, 69k, 66k, and 62kDa fragments. The microsequence analysis revealed that the 69k and 66kDa components from the 10 degrees C-acclimated carp contained an N-terminal amino acid sequence different from that of 62kDa. The four fragments from the 30 degrees C-acclimated carp showed the same sequence as that of the 69k and 66kDa components from the 10 degrees C-acclimated carp, except that the 2nd amino acid, Ala, of the 10 degrees C-acclimated LMM was replaced by Thr. DNA fragments encoding an N-terminal region of LMM were amplified by PCR or reverse transcriptase-PCR, demonstrating that the two acclimated groups further contained several amino acids substituted.
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PMID:Temperature acclimation induces light meromyosin isoforms with different primary structures in carp fast skeletal muscle. 788 20

In Drosophila and Caenorhabditis, signal transduction pathways initiated by the activation of receptor-protein tyrosine kinases can mediate developmental fate decisions. In order to examine whether similar mechanisms are employed during mammalian embryogenesis, we undertook a search for novel protein kinases expressed during heart development in the mouse. The primitive mouse heart is formed between 7.75 and 8.5 days post coitum (dpc) and consists of myocardial and endocardial cells. A reverse transcriptase polymerase chain reaction-based approach was used to amplify protein kinase specific products from cDNAs obtained from 8.5 dpc heart tissue. Twenty independent PCR products corresponding to either protein serine/threonine or tyrosine kinases were identified. In this report, we describe the characterization of two of the genes corresponding to the novel PCR products (designated Hek2 and msk). Hek2 encodes the mouse ortholog of human HEK2, a recently identified member of the eph receptor-protein tyrosine kinase gene family. Prior to and at the time of heart formation (7.5-8.0 dpc), Hek2 is expressed in the cranial (rostral) region of the embryo from which a subpopulation of cells will give rise to the rudimentary heart. Between 8.0 and 9.5 dpc, Hek2 mRNA expression is observed in myocardial cells, head mesenchyme and paraxial mesoderm. Hek2 transcripts are not detected in endocardial cells. After 9.5 dpc, Hek2 expression is downregulated. msk (for myocardial SNF1-like kinase) encodes a putative protein serine/threonine kinase most similar to the yeast gene SNF1. msk mRNA expression is restricted to myocardial cells and their progenitors in the 7.75-8.5 dpc developing heart. Subsequently, msk mRNA expression is rapidly downregulated. The patterns of Hek2 and msk expression suggest that these protein kinases may function during development of the primitive heart.
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PMID:Identification of novel protein kinases expressed in the myocardium of the developing mouse heart. 789 99

We have determined the DNA sequence of the mitochondrial plasmid from Neurospora intermedia strain Fiji N6-6. The plasmid contains a 1278-codon open reading frame that is 49% identical to the open reading frame of the mitochondrial plasmid from the LaBelle strain of N. intermedia, which is known to encode a DNA-dependent DNA polymerase. The results of polymerase assays and photolabeling studies, the high degree of identity with the LaBelle plasmid polymerase, and the observation that the Fiji polymerase activity in a reaction utilizing endogenous template is not affected by removal of RNA suggest that the Fiji plasmid also encodes a DNA-dependent DNA polymerase. Comparison of regions of amino acids that are highly conserved in the two plasmid polymerases to family B polymerases reveals good correlates for the three major polymerase motifs and suggests that previously identified motifs characteristic of reverse transcriptase found in the LaBelle sequence are not significant. The polymerases encoded by the Fiji and LaBelle plasmids are unusual in that the amino acid sequence Asp-Thr-Asp, which forms the core of the third motif in family B polymerases, is not present in either Fiji or LaBelle. A version of the motif containing Thr-Thr-Asp exists in both sequences.
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PMID:Two Neurospora mitochondrial plasmids encode DNA polymerases containing motifs characteristic of family B DNA polymerases but lack the sequence Asp-Thr-Asp. 848 47

The characterization of naturally occurring mutations is one way to approach functionally significant domains of polypeptides. About 10 mutations have been reported in factor XIII (FXIII) A-subunit deficiency, but very little is known about the effects of the mutations on the expression or the structure of this enzyme. In this study, the recent crystallization of FXIII A-subunit and determination of the three-dimensional model were used for the first time to pursue the structural consequences of mutations in the A-subunit. The molecular analysis of four families from Sweden, Germany, and Denmark revealed four previously unreported point mutations. Three of the mutations were missense mutations, Arg326-->Gln, Arg252-->Ile, and Leu498-->Pro, and one was a nonsense mutation, a deletion of thymidine in codon for Phe8 resulting in early frameshift and premature termination of the polypeptide chain. In the case of the nonsense mutation, delT Phe8, the steady-state mRNA level of FXIII A-subunit was reduced, as quantitated by reverse transcriptase-polymerase chain reaction and solid-phase minisequencing. In contrast, none of the missense mutations affected mRNA levels, indicating the possible translation of the mutant polypeptides. However, by enzyme-linked immunosorbent analysis and immunofluorescence, all the patients demonstrated a complete lack of detectable factor XIIIA antigen in their platelets. In the structural analysis, we included the mutations described in this work and the Met242-->Thr mutation reported earlier by us. Interestingly, in the three-dimensional model, all four missense mutations are localized in the evolutionarily conserved catalytic core domain. The substitutions are at least 15 A away from the catalytic cleft and do not affect any of the residues known to be directly involved in the enzymatic reaction. The structural analyses suggest that the mutations are most likely interfering with proper folding and stability of the protein, which is in agreement with the observed absence of detectable FXIIIA antigen. Arg326, Arg252, and Met242 are all buried within the molecule. The Arg326-->Gln and Arg252-->Ile mutations are substitutions of smaller, neutral amino acids for large, charged residues. They disrupt the electrostatic balance and hydrogen-bonding interactions in structurally significant areas. The Met242-->Thr mutation is located in the same region of the core domain as the Arg252-->Ile site and is expected to have a destabilizing effect due to an introduction of a smaller, polar residue in a tightly packed hydrophobic pocket. The substitution of proline for Leu498 is predicted to cause unfavorable interatomic contacts and a disruption of the alpha-helix mainchain hydrogen-bonding pattern; it is likely to form a kink in the helix next to the dimer interface and is expected to impair proper dimerization of the A-subunits. In the case of all four missense mutations studied, the knowledge achieved from the three-dimensional model of crystallized FXIII A-subunit provides essential information about the structural significance of the specific residues and aids in understanding the biologic consequences of the mutations observed at the cellular level.
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PMID:Four novel mutations in deficiency of coagulation factor XIII: consequences to expression and structure of the A-subunit. 854 36

It has been reported recently that the human foamy virus (HFV) Pol polyprotein of 120 kDa is synthesized in the absence of the active HFV aspartic protease. To gain more information on how the 120-kDa Pro-Pol protein is synthesized, mutant HFV genomes were constructed and the resulting proviruses were analyzed with respect to HFV pol expression and infectivity. HFV proviruses that contain termination codons in the nucleocapsid domain of gag and thus lack a gag-pol overlap region assumed to be required for translational frameshifting, nevertheless expressed the 120-kDa Pro-Pol precursor, the 80-kDa reverse transcriptase/RNase H, and a 40-kDa integrase in amounts similar to those observed for wild-type genomes. Since a Gag-independent expression of authentic Pol proteins was detectable in cells transfected with eukaryotic HFV pol expression plasmids, the data indicate that the HFV Pol precursor of 120 kDa is expressed independently of Gag by a mechanism that does not rely on ribosomal frameshifting, since the postulated HFV Gag-Pol protein of 190 kDa was not detectable under the conditions used. Furthermore, replacement of the Met residue by Thr at position 9 in pol within the gag-pol overlap region resulted in strongly reduced HFV Pol polyprotein expression and infectivity of the resulting proviruses. This Met residue of pol conserved in foamy virus sequences is the likely candidate for translational initiation of the 120-kDa Pro-Pol polyprotein. trans complementation of the HFV mutant with the Met-to-Thr substitution in the pol gene by a eukaryotic plasmid that expressed the HFV Pro-Pol protein resulted in partial recovery of infectivity. When HFV pol was fused in frame to gag, an engineered 190-kDa Gag-Pol fusion protein was formed and the enzymatic activity of the HFV protease was partially retained. The results imply that HFV is the first retrovirus that expresses a Pol polyprotein without formation of a Gag-Pol fusion protein.
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PMID:The human foamy virus pol gene is expressed as a Pro-Pol polyprotein and not as a Gag-Pol fusion protein. 855 61

Human membrane cofactor protein (MCP, CD46) is a receptor for the measles virus and serves as a complement regulator which protects host cells from autologous complement attack. MCP is highly polymorphic due to a variety of mRNA splice products. The levels of MCP expression on T and myeloid cell lines are usually two-eightfold higher than those on their normal counterparts, whereas Burkitt's lymphoma B cell lines express less MCP than B cell lineages carrying no EB virus. The molecule has a Ser/Thr-rich (ST) domain adjacent to the functional domain, namely short consensus repeats (SCR). The ST domain and a cytoplasmic tail (CYT) contribute to the MCP polymorphism. The ST domain is encoded by three exons (A, B and C) and major ST isoforms are STABC, STBC and STC. The authors investigated the relationship between the expression levels and isoform usage of MCP by flow cytometry using specific antibodies against STA and STC, by reverse transcriptase-polymerase chain reaction (RT-PCR) with size markers for each splice variant, and by RT-PCR/Southern blotting using a specific probe for STA. The results were (1) the profiles of mean shifts of myeloid and T cell lines were STC < STA on flow cytometry while those of B cell lines and normal blood cells were STA < STC; (2) all cell lines tested by RT-PCR expressed the messages for the isoforms STBC/CYT1, STC/CYT1, STBC/CYT2, and STC/CYT2. The band for STABC/CYT2 overlapped that for STC/CYT1, and the band for STABC/CYT1 was marginal in all cell lines examined; (3) semi-quantitative analysis of the STABC isoforms by Southern blotting indicated the presence of high levels of the STABC messages in myeloid and T-cell lines in comparison with B lymphoid cells and normal leucocytes. Thus, the quantity of MCP expressed parallels the STABC message level, which is up-regulated in T and myeloid leukaemia cell lines.
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PMID:High expression of membrane cofactor protein of complement (CD46) in human leukaemia cell lines: implication of an alternatively spliced form containing the STA domain in CD46 up-regulation. 855 81

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated (as single agents or in combination) with (minus)-2', 3'-dideoxy-3'-thiacytidine (3TC) and the following HIV-1-specific non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs): 2', 5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5'-(4'-amino-1',2'-oxathi ole)-2',2'-dioxide derivative of 3-methylthymidine (TSAO-m3T), the thiocarboxanilides UC10 and UC42, bis(heteroaryl)piperazine (BHAP) derivative U90152, and the 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivative 5-isopropyl-1-ethoxymethyl-6-benzyluracil (MKC-442). When used individually, the compounds led to the emergence of HIV-1 strains containing the following mutations in the RT: Glu138 to lysine for TSAO-m3T, Met184 to valine for 3TC, Lys103 to threonine/asparagine for the thiocarboxanilides, and Tyr181 to cysteine for BHAP and MKC-442. When 3TC was combined with TSAO-m3T, UC10, UC42, BHAP, or MKC-442, breakthrough of virus was markedly delayed or even suppressed. For these drug combinations, the concentrations of the individual drugs could be lowered by > or = 25-50-fold to suppress virus breakthrough compared with the individual use of the compounds. The concomitant presence of the Lys138 and Ile/Val184 mutations was found in the RT of the mutant viruses that emerged with combination therapy of the lowest concentrations of 3TC with either the lowest concentrations of TSAO-m3T or UC10 (approximately 0.5-3-fold the EC50 value). These virus strains retained high sensitivity to other NNRTIs such as BHAP or HEPT. The virus mutants that arose in the presence of combinations of the lowest concentrations of 3TC with either BHAP or HEPT predominantly contained the Cys181 mutation in the RT. In one case, the Ile181 mutation was found. The latter mutations, particularly the Ile181 mutation, resulted in markedly decreased sensitivity to the NNRTIs but not to 3'-azido-2', 3'-dideoxythymidine or 3TC.
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PMID:Marked inhibitory activity of non-nucleoside reverse transcriptase inhibitors against human immunodeficiency virus type 1 when combined with (-)2',3'-dideoxy-3'-thiacytidine. 862 38

Congenital methemoglobinemia caused by an erythrocytic deficiency of cytochrome b5 reductase (b5R; type I) in African-American individuals was first reported by this laboratory. The rarity of this observation is possibly due to the difficulty detecting cyanosis that is masked by naturally occurring dark skin pigment. Since previous biochemical studies on the African-American family with variant enzyme b5R-Shreveport showed enzyme instability, we focused on molecular analysis of its transcript. The transcript size was the same as that of a normal control. The nucleotide sequence of both normal and variant transcripts were examined by directly sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) product. The propositus was found to be homozygous for a G to A transition at codon 212 in exon 8, changing a glutamate to a lysine (E212K). In addition, a C to G transversion was found at codon 116 in exon 5, changing a threonine to a serine (T116S). Using allele-specific PCR, we determined that E212K was found only in the propositus and her heterozygous mother. Furthermore, E212K is predicted to disrupt an alpha-helix peptide structure of b5R, suggesting that this is likely the disease-causing mutation. In contrast, T116S was found to be a high-frequency polymorphism specific for the African-American population. The E212K mutation is uniquely present in the 3' end of the b5R gene (exon 8), which differs from those b5R mutations found among Japanese subjects (exons 3 and 5) and in an Italian subject (exon 4) and, thus, further contributes to our understanding of the structure/function relationship of this housekeeping enzyme.
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PMID:A novel mutation found in the 3' domain of NADH-cytochrome B5 reductase in an African-American family with type I congenital methemoglobinemia. 863 21

The SPOC1 cell, a novel goblet cell line derived from rat trachea, was tested for its ability to exhibit regulated mucin secretion in response to purinergic (P2) agonists. High-molecular mass glycoconjugates (HMMGs) purified by CsCl-density-gradient centrifugation had a buoyant density of 1.45 g/ml. The purified HMMG material exhibited a single major band with an apparent molecular mass of greater than 1000 kDa in SDS/ polyacrylamide gels stained with silver or blotted and stained with soya-bean agglutinin. [3H]HMMG was resistant to proteoglycan-degrading enzymes, but was susceptible to neuraminidase. The HMMG was approx. 91% carbohydrate by weight, and the glycosides were O-linked. The HMMG amino acid composition was enriched in Ser and Thr (sum 27%). Thus SPOC1-cell HMMG possess the characteristics of mucin. Mucin secretion by SPOC1 cells, grown on permeable supports and perfused luminally, was stimulated by ATP, UTP and adenosine 5'-[gamma-thio]triphosphate (100 microM) 4-5-fold over a baseline of 4 ng/min. The three dose-effect relations were nearly identical (K0.5 approximately 4 microM). SPOC1 cells grown on plastic and rat tracheal epithelial primary cells responded similarly to ATP and/or UTP. SPOC1 cells failed to respond to other purinergic agonists, either luminally or serosally, and consequently seem to possess an apical membrane P2u purinoceptor. SPOC1-cell total RNA was probed for P2u purinoceptor mRNA. Using conserved primers for both reverse transcriptase and PCR, a single band of the predicted size was observed, which had a nucleotide base sequence identical with the rat P2u purinoceptor mRNA. Thus SPOC1 cells secrete mucin under the control of a P2u purinoceptor; they should prove useful in dissecting the associated cellular regulatory pathways.
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PMID:P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways. 867 Jan 74

We report here the cloning of a new member of the endothelium differentiation gene (edg) subfamily of G-protein-coupled receptors. This novel cDNA sequence was cloned from the ovine pars tuberalis using a reverse transcriptase polymerase chain reaction (RT-PCR) amplification with degenerate primers homologous to the highly conserved II and VII transmembrane domains of the G-protein coupled receptor gene family. The PCR product was random primed with 32P and used as a probe to screen a size-selected cDNA ovine pars tuberalis library, which resulted in the isolation of a single clone of 2700 bp. This novel sequence was named edg-2, because its nucleic acid sequence was 55% homologous over 501nt overlap to an orphan sequence cloned from human endothelial cells, the endothelial differentiation gene, edg-1. The highest degree of aminoacid homology (42%) occurs in the seven putative transmembrane domains, particularly between the transmembrane domains III and VI (53% and 64%, respectively). The intervening hydrophilic domains are short and there are numerous putative phosphorylation sites for Ser/Thr-protein kinases in the second and third intracellular loop and in the COOH-terminal domain. Through Northern analysis of total RNA, low levels of at least four transcripts of 2.3, 2.5, 3.2 and 4 kb were found in sheep cerebral cortex and a 4.2 kb transcript was observed in NIH/3T3 fibroblasts. In addition, edg-2 transcripts (415 bp) were amplified by RT-PCR from pars tuberalis, cerebral blood vessels, hypothalamus, and retina. Serum stimulation of Chinese hamster ovary (CHO) cells expressing the edg-2 receptor resulted in increased cell proliferation, as measured by [3H]-thymidine incorporation. Edg-1 and edg-2 appear to be distinct genes that may encode protein products that bind the same or related ligand.
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PMID:Cloning and characterization of a new member of the G-protein coupled receptor EDG family. 883 98


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