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N Nelson

Publications and source records attributed to N Nelson.

At least 127 records · Page 7Linked to original sources
PubMed1993

Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferon-induced gene transcription.

We previously isolated a cDNA clone encoding interferon consensus sequence-binding protein (ICSBP), a member of the interferon regulatory factor (IRF) family, that binds to the interferon (IFN)-stimulated response element (ISRE) of many IFN-regulated genes. In this investigation, we studied the functional role of ICSBP by transient cotransfection of ICSBP cDNA with IFN-responsive reporter genes into the human embryonal carcinoma cell line N-Tera2. These cells were shown not to express ICSBP or IRF-2, thus allowing functional analysis of transfected cDNAs. Cotransfection of ICSBP into cells treated with retinoic acid or any of the IFNs (alpha, beta, or gamma) repressed expression of a chloramphenicol acetyltransferase reporter driven by the major histocompatibility complex class I gene promoter. Similarly, ICSBP repressed expression of chloramphenicol acetyltransferase reporters driven by the ISREs of the 2'-5' oligoadenylate synthetase, guanylate-binding protein, and ISG-15 genes in IFN-treated cells. The repression was dependent on the presence of the ISRE in the reporter. Deletion analysis showed that the putative N-terminal DNA binding domain of ICSBP by itself is capable of mediating the repression. Using the same cotransfection conditions as for ICSBP, a similar repression of these reporters was observed with IRF-2. Finally, ICSBP repressed the IRF-1-mediated induction of major histocompatibility complex class I and IFN-beta reporters in the absence of IFN or retinoic acid. Taken together, these results suggest that ICSBP is a negative regulatory factor capable of repressing transcription of target genes induced by IFN, retinoic acid, or IRF-1.

Base Sequence↗
PubMed1992

Cloning and expression of a cDNA encoding the transporter of taurine and beta-alanine in mouse brain.

A taurine/beta-alanine transporter was cloned from a mouse brain cDNA library by screening with a partial cDNA probe of the glycine transporter at low stringency. The deduced amino acid sequence predicts 590 amino acids with typical characteristics of the sodium-dependent neurotransmitter transporters such as sequence homology and membrane topography. However, the calculated isoelectric point of the taurine/beta-alanine transporter is more acidic (pI = 5.98) than those (pI > 8.0) of other cloned neurotransmitter transporters. Xenopus oocytes injected with cRNA of the cloned transporter expressed uptake activities with Km = 4.5 microM for taurine and Km = 56 microM for beta-alanine. Northern hybridization showed a single transcript of 7.5 kilobases that was highly enriched in kidney and distributed evenly in various parts of the brain. In situ hybridization showed the mRNA of the taurine/beta-alanine transporter to be localized in the corpus callosum, striatum, and anterior commisure. Specific localization of the taurine/beta-alanine transporter in mouse brain suggests a potential function for taurine and beta-alanine as neurotransmitters.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of the bovine cardiac sodium-calcium exchanger.

Two clones (p17 and p13), each containing the complete coding sequence for the bovine cardiac Na+/Ca2+ exchanger, were obtained from a lambda gt10 cDNA library by screening with cDNA probes from the canine exchanger. The coding sequence of clone p17 was 92 and 98% identical to the canine cDNA at the nucleotide and amino acid levels, respectively. Nine of the 21 amino acid differences between the two exchangers were found within the 32-amino acid signal sequence. The sequenced portions of the 3' untranslated regions of the cow and dog clones were 88% identical. Na+/Ca2+ exchange activity was expressed in Xenopus laevis oocytes injected with cRNA from clone p17, and in COS cells transfected with expression vectors containing p17. Immunoprecipitation of 35S-labeled proteins from transfected cells with an antibody against the N-terminal portion of the bovine exchanger showed the presence of a 120-kDa protein corresponding to the intact cardiac exchanger. The second bovine clone (p13) did not express exchange activity in either of the above expression systems, presumably because it contained a 300-bp insert with multiple stop codons which interrupted the coding sequence. Comparison of the 5' untranslated regions of p13 and p17 revealed a 156-bp segment in p17 that was apparently spliced out of p13. This segment contained a short open reading frame. A chimera encoding the 5' untranslated region of p13 and the coding sequence of p17 exhibited only a modest (74%) increase in expressed exchange activity in transfected cells compared to p17, suggesting that the presence of the upstream open reading frame in p17 did not greatly reduce translation efficiency. The results suggest that alternate splicing mechanisms may be involved in processing mRNA for the bovine cardiac exchanger.

Amino Acid Sequence↗
PubMed1992

Expression of a mouse brain cDNA encoding novel gamma-aminobutyric acid transporter.

A nipecotic acid-resistant gamma-aminobutyric acid (GABA) transporter was cloned from a mouse brain cDNA library. The 2.3-kilobase cDNA clone contains an open reading frame of 1842 nucleotides encoding a protein of 614 amino acids. The predicted amino acid sequence indicates it is a member of the gene family of the sodium-dependent neurotransmitter transporters. The new GABA transporter, named GAT2, is highly homologous to the betaine transporter (BGT1) cloned from canine kidney. However, GAT2 expression in the brain distinguished it from BGT1 which was exclusively expressed in the kidney. The transcripts of GAT2 were found in the cerebral cortex, cerebellum, and brainstem as well as in kidney. Expression of GAT2 in Xenopus oocytes revealed a Km of 79 microM for GABA uptake which is about 10-fold higher than that of the high affinity GABA transporter (GAT1). The pharmacology of GAT2 is different from that of GAT1 because of lack of inhibition by guvacine and nipecotic acid and sensitivity to high concentrations of betaine and beta-alanine. GAT2 transports betaine with a Km of about 200 microM, but no significant transport of beta-alanine could be detected. The presence of mRNA encoding GAT2 in parts of the brain suggests it is a neurotransmitter transporter.

Amino Acid Sequence↗
PubMed1992

Photosynthetic reaction center genes in green sulfur bacteria and in photosystem 1 are related.

Oxygenic photosynthesis of chloroplasts and cyanobacteria involves two photosystems, which originate from different prokaryotic ancestors. The reaction center of photo-system 2 (PS2) is related to the well-characterized reaction center of purple bacteria, while the reaction center of photosystem 1 (PS1) is related to the green sulfur bacteria, as is convincingly documented here. An operon encoding the P840 reaction center of Chlorobium limicola f.sp. thiosulfatophilum has been cloned and sequenced. It contains two structural genes, coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the PS1 reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS1, and for the acceptors A0, A1, and FeS center X are conserved. The second protein is related to the PS1 subunit carrying the FeS centers A and B. An adjacent third gene, not belonging to the reaction center, encodes a protein related to dolichyl-phosphate-D-mannose synthase from yeast. The different origins of PS1 and PS2 are discussed.

Amino Acid Sequence↗
PubMed1992

The photosystem I-like P840-reaction center of green S-bacteria is a homodimer.

An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.

Amino Acid Sequence↗
PubMed1992

A family of genes encoding neurotransmitter transporters.

The genomic and cDNA clones of the mouse gamma-aminobutyric acid transporter were sequenced and analyzed. The genomic clone contains 12 introns including 1 intron prior to the initiator methionine. The second intron comes immediately after the stretch of amino acids that is most conserved among the neurotransmitter transporters sequenced so far. By using a probe constructed according to this conserved region, several partial genomic clones were isolated. Sequence analysis of those clones reveals not only homology to the family of neurotransmitter transporters within the reading frame but also an identical location of an exon-intron junction after the conserved region. A search of the GenBank data base (April 1991) revealed that two invertebrate genes exhibit homology to the conserved sequence of the above family. One, a Drosophila melanogaster gene, encoded the N-terminal part of a protein homologous to neurotransmitter transporters and the second was in Caenorhabditis elegans. The Drosophila gene contains an intron that starts at a position identical to the corresponding positions of all the mammalian genes of the family.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of a glycine transporter from mouse brain.

We have isolated a cDNA clone from a mouse brain library encoding the glycine transporter (GLYT). Xenopus oocytes injected with a synthetic mRNA accumulated [3h]glycine to levels of up to 80-fold above control values. The uptake was specific for glycine and dependent on the presence of Na+ and Cl- in the medium. The cDNA sequence predicts a highly hydrophobic protein of 633 amino acids with 12 potential transmembrane helices. The predicted amino acid sequence has 40-45% identity to the GABA, noradrenaline, serotonin and dopamine transporters. This implies that all of these neurotransmitter transporters may have evolved from a common ancestral gene that diverged into the GABA, glycine and catecholamine subfamilies at nearly the same time.

Amino Acid Sequence↗
PubMed1992

Evolution of organellar proton-ATPases.

Proton ATPases function in biological energy conversion in every known living cell. Their ubiquity and antiquity make them a prime source for evolutionary studies. There are two related families of H(+)-ATPases; while the family of F-ATPases function in eubacteria chloroplasts and mitochondria, the family of V-ATPases are present in archaebacteria and the vacuolar system of eukaryotic cells. Sequence analysis of several subunits of V- and F-ATPases revealed several of the important steps in their evolution. Moreover, these studies shed light on the evolution of the various organelles of eukaryotes and suggested some events in the evolution of the three kingdoms of eubacteria, archaebacteria and eukaryotes.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of the interleukin-1 beta converting enzyme.

Interleukin-1 beta (IL-1 beta) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor. A complementary DNA encoding a protease that carries out this cleavage has been cloned. Recombinant expression in COS-7 cells enabled the cells to process precursor IL-1 beta to the mature form. Sequence analysis indicated that the enzyme itself may undergo proteolytic processing. The gene encoding the protease was mapped to chromosomal band 11q23, a site frequently involved in rearrangement in human cancers.

Amino Acid Sequence↗
PubMed1992

Compartment acidification is required for efficient sorting of proteins to the vacuole in Saccharomyces cerevisiae.

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles. These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles. The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing. Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH. A similar type of missorting defect can be induced in wild-type cells at pH 7.5. These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.

Aspartic Acid Endopeptidases↗
PubMed1992

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.

Amino Acid Sequence↗
PubMed1992

Structural conservation and functional diversity of V-ATPases.

The vacuolar system of eukaryotic cells contains a large number of organelles that are primary energized by an H(+)-ATPase that was named V-ATPase. The structure and function of V-ATPases from various sources was extensively studied in the last few years. Several genes encoding subunits of the enzyme were cloned and sequenced. The sequence information revealed the relations between V-ATPases and F-ATPases that evolved from common ancestral genes. The two families of proton pumps share structural and functional similarity. They contain distinct peripheral catalytic sectors and hydrophobic membrane sectors. Genes encoding subunits of V-ATPase in yeast cells were interrupted to yield mutants that are devoid of the enzyme and are sensitive to pH and calcium concentrations in the medium. The mutants were used to study structure, function, molecular biology, and biogenesis of the V-ATPase. They also shed light on the functional assembly of the enzyme in the vacuolar system.

Amino Acid Sequence↗
PubMed1992

Organellar proton-ATPases.

Proton pumps that belong to the families of F-ATPases and V-ATPases operate without the formation of a phosphorylated intermediate and contain several subunits grouped into distinct catalytic and membrane sectors. Recent studies on the structure and molecular biology of V-ATPases shed light not only on the structure-function relations between the two families, but also on their evolution in all organisms.

Amino Acid Sequence↗
PubMed1992

Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35.

Two human cDNA clones that encode different interleukin-8 (IL8) receptors have recently been isolated. The interleukin-8 receptor type 1 (IL8R1) binds IL8 only, whereas the interleukin-8 receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an interleukin-8 receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.

Amino Acid Sequence↗
PubMed1992

Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes.

Photosystem I contains several peripheral membrane proteins that are located on either positive (luminal) or negative (stromal or cytoplasmic) sides of thylakoid membranes of chloroplasts or cyanobacteria. Incorporation of two peripheral subunits into photosystem I of the cyanobacterium Synechocystis species PCC 6803 was studied using a reconstitution system in which radiolabeled subunits II (PsaD) and IV (PsaE) were synthesized in vitro and incubated with the isolated thylakoid membranes. After such incubation, the subunits were found in the membranes and were resistant to digestion with proteases and removal by 2 molar NaBr. All of the radioactive proteins incorporated in the membrane were found in the photosystem I complex. The subunit II was assembled specifically into cyanobacterial thylakoid membranes and not into Escherichia coli cell membranes or thylakoid membranes isolated from spinach. The assembly process did not require ATP or proton motive force, and it was not stimulated by ATP. The assembly of subunits II and IV into thylakoid membranes isolated from the strain AEK2, which lacks the gene psaE, was increased two- to threefold. The incorporation of subunit II was 15 to 17 times higher in the thylakoids obtained from the strain ADK3 in which the gene psaD has been inactivated. However, assembly of subunit IV in the same thylakoids was reduced by 65%, demonstrating that the presence of subunit II is required for the stable assembly of subunit IV. Large deletions in subunit II prevented its incorporation into thylakoids and assembly into photosystem I, suggesting that the overall conformation of the protein rather than a specific targeting sequence is required for its assembly into photosystem I.

Journal Article↗
PubMed1992

Structure and function of V-ATPases in endocytic and secretory organelles.

Chromaffin granules and clathrin-coated vesicles are major sources for V-ATPases of mammalian cells. Studies of these organelles have helped us to understand the structure and function of the enzyme. It was shown that V-ATPases are composed of distinct catalytic and membrane sectors containing several subunits. The subunit stoichiometry was determined to be 3A, 3B, 1C, 1D, 1E, 6c (proteolipids), 1Ac115 and ?Ac39. Additional subunits are likely to be discovered. Resolution and reconstitution of the enzyme revealed that the catalytic and membrane sectors are interdependent for their partial activity. The catalytic sector has no ATPase activity when detached from the membrane sector, and the membrane sector when depleted of the catalytic sector does not conduct protons. The mechanistic significance of these properties is discussed.

Adenosine Triphosphatases↗