Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase.

ABSTRACT: A partial cDNA clone coding for the haem-binding domain of NADH:nitrate reductase (EC 1.6.6.1) (NR) from the unicellular green alga Chlorella vulgaris has been isolated, sequenced and expressed. A 1.2 kb cDNA (pCVNR1) was isolated from a lambda gt11 expression library produced from polyadenylated RNA extracted from nitrate-grown Chlorella cells. pCVNR1 hybridized to a 3.5 kb mRNA transcript that was nitrate-inducible and absent from ammonium-grown cells. The entire sequence of pCVNR1 was obtained and found to have a single uninterrupted reading frame. The derived amino acid sequence of 318 amino acids has a 45-50% similarity to higher-plant NRs, including Arabidopsis thaliana, spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum). A comparison with the putative domain structure of higher-plant nitrate reductases suggested that this sequence contains the complete haem-binding domain, approximately one-third of the Mo-pterin domain and no FAD-binding domain. A 32% sequence similarity is evident when comparing the Chlorella NR haem domain with that of calf cytochrome b5. Expression of pCVNR1 in a pET vector synthesized a 35 kDa protein that was antigenic to anti-(Chlorella NR) antibody. The spectral properties of this protein (reduced and oxidized) in the 400-600 nm region are identical with those of native Chlorella NR and indicate that haem is associated with the protein.

SUBMITTER: Cannons AC

PROVIDER: S-EPMC1151469 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin.

Project description:A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phosphoprotein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A beta-galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3' noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain approximately 12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain.

| S-EPMC282646 | biostudies-other

cDNA clone encoding Drosophila transcription factor TFIID.

Project description:Proper initiation of transcription by RNA polymerase II requires the TATA-consensus-binding transcription factor TFIID. A cDNA clone encoding the Drosophila TFIID protein has been isolated and characterized. The deduced amino acid sequence reveals an open reading frame of 353 residues. The carboxyl-terminal 180 amino acids are approximately 80% identical to yeast TFIID and 88% identical to human TFIID. The amino-terminal portions of the yeast and Drosophila TFIID proteins lack appreciable homology, whereas the Drosophila and human amino termini appear qualitatively similar. In addition, the amino-terminal region of the Drosophila TFIID contains several sequence motifs that are found in other Drosophila proteins which appear to regulate transcription.

| S-EPMC55121 | biostudies-other

A new strategy to produce a defensin: stable production of mutated NP-1 in nitrate reductase-deficient Chlorella ellipsoidea.

Project description:Defensins are small cationic peptides that could be used as the potential substitute for antibiotics. However, there is no efficient method for producing defensins. In this study, we developed a new strategy to produce defensin in nitrate reductase (NR)-deficient C. ellipsoidea (nrm-4). We constructed a plant expression vector carrying mutated NP-1 gene (mNP-1), a mature ?-defensin NP-1 gene from rabbit with an additional initiator codon in the 5'-terminus, in which the selection markers were NptII and NR genes. We transferred mNP-1 into nrm-4 using electroporation and obtained many transgenic lines with high efficiency under selection chemicals G418 and NaNO(3). The mNP-1 was characterized using N-terminal sequencing after being isolated from transgenic lines. Excitingly, mNP-1 was produced at high levels (approximately 11.42 mg/l) even after 15 generations of continuous fermentation. In addition, mNP-1 had strong activity against Escherichia coli at 5 µg/ml. This research developed a new method for producing defensins using genetic engineering.

| S-EPMC3557228 | biostudies-literature

Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance.

Project description:Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.). Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influence on developmental and protective abilities of plant against biotic stress. The transgenic lines of CsDFR and CsANR produced early flowering and better seed yield. Both types of transgenic tobacco showed higher content of flavonoids than control. Flavan-3-ols such as catechin, epicatechin and epicatechingallate were found to be increased in transgenic lines. The free radical scavenging activity of CsDFR and CsANR transgenic lines was improved. Oxidative stress was observed to induce lesser cell death in transgenic lines compared to control tobacco plants. Transgenic tobacco overexpressing CsDFR and CsANR also showed resistance against infestation by a tobacco leaf cutworm Spodoptera litura. Results suggested that the overexpression of CsDFR and CsANR cDNA in tobacco has improved flavonoids content and antioxidant potential. These attributes in transgenic tobacco have ultimately improved their growth and development, and biotic stress tolerance.

| S-EPMC3688816 | biostudies-literature

Characterization of a cDNA clone encoding the visna virus transactivating protein.

Project description:The unique pathogenesis of lentiviral infections in humans and ruminant animals may be explained, in part, by the complex mechanisms regulating transcription and translation of their viral genes. This report demonstrates that a visna virus-encoded protein transactivates viral gene expression. A 1.4-kilobase cDNA clone encodes two distinct proteins with apparent molecular masses of 21.5 and 10 kDa. We demonstrate that the 10-kDa species is the visna virus transactivating (Tat) protein; the other species may be analogous to the rev (formerly art or trs) gene product of human immunodeficiency virus.

| S-EPMC286480 | biostudies-other

cDNA clone and expression analysis of ?-Tropomyosin during Japanese flounder (Paralichthys olivaceus) metamorphosis.

Project description:Tropomyosin (TM) plays a critical role in skeletal and cardiac muscle development and function. To assess the functional significance of ?-TM in Japanese flounder (Paralichthys olivaceus) development and metamorphosis, cDNA from Japanese flounder was cloned and ?-TM mRNA measured during development and metamorphosis. The full-length cDNA is 1 191 bp, including a 5'-untranslated region of 114 bp, a 3'-UTR of 222 bp, and an open reading frame of 855 bp encoding a polypeptide of 284 amino acids. Real-time quantitative PCR revealed that ?-TM mRNA is initially expressed in unfertilized ovum, indicating the ?-TM gene is maternal. Relatively low mRNA levels were observed in different embryonic stages. A higher level of ?-TM mRNA was detected 3 days post hatching (dph), while the highest level was measured at 29 dph (metamorphic climax) after which it declined towards the end of metamorphosis. The expression of ?-TM mRNA was up-regulated in thyroid hormone-treated larvae at 36 dph, but there was no marked difference at other stages when compared to control animals. After thiourea treatment, the expression of ?-TM mRNA declined slightly. These data provide basic information that can be utilized in further studies into the role of ?-TM in P. olivaceus development and metamorphosis.

| S-EPMC5031678 | biostudies-literature

Thioredoxin reductase regulates the induction of haem oxygenase-1 expression in aortic endothelial cells.

Project description:Certain selenoproteins such as GPX-1 (glutathione peroxidase-1) and TrxR1 (thioredoxin reductase-1) possess important antioxidant defence functions in vascular endothelial cells. Reduced selenoprotein activity during dietary selenium (Se) deficiency can result in a compensatory increase of other non-Se-dependent antioxidants, such as HO-1 (haem oxygenase-1) that may help to counteract the damaging effects of oxidant stress. However, the role of individual selenoproteins in regulating vascular-derived protective gene responses such as HO-1 is less understood. Using an oxidant stress model based on Se deficiency in BAECs (bovine aortic endothelial cells), we sought to determine whether TrxR1 activity may contribute to the differential regulation of HO-1 expression as a function of altered redox environment. Se-sufficient BAECs up-regulated HO-1 expression following stimulation with the pro-oxidant, 15-HPETE (15-hydroperoxyeicosatetraenoic acid), and levels of this antioxidant inversely correlated with EC apoptosis. While Se-deficient BAECs exhibited higher basal levels of HO-1, it was not up-regulated upon 15-HPETE treatment, which resulted in significantly higher levels of pro-apoptotic markers. Subsequent results showed that HO-1 induction depended on the activity of TrxR1, as proved with chemical inhibitor studies and direct inhibition with TrxR1 siRNA. Finally, restoring intracellular levels of the reduced substrate Trx (thioredoxin) in Sedeficient BAECs was sufficient to increase HO-1 activation following 15-HPETE stimulation. These data provide evidence for the involvement of the Trx/TrxR system, in the regulation of HO-1 expression in BAECs during pro-oxidant challenge.

| S-EPMC1386018 | biostudies-literature

Functional expression of a cDNA encoding a human ecto-ATPase.

Project description:1 The metabolism of extracellular nucleotides plays an important role in nucleotide signalling mediated by P2 receptors. The nucleotide sequence encoding a putative human ecto-ATPase named CD39L1 was reported recently. However, the biological activity of this protein has not been established. 2 Based on the sequence of CD39L1 we isolated from mRNA from human ECV-304 cells a sequence encoding a 495 amino acid protein that is identical to CD39L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequence of a genomic DNA clone indicates that the CD39L1 gene corresponds to an alternative spliced form of the human ecto-ATPase. 3 Stable expression of isolated sequence in NIH-3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto-ATPase. 4 The human ecto-ATPase hydrolyzed all naturally occurring nucleoside triphosphates in a Ca(2+)- or Mg(2+)-dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosphates. The apparent Km and Vmax values were: 394+/-62 microM and 107+/-7 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ATP, and 102+/-33 microM and 4+/-0.4 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ADP, respectively. 5 In conclusion, we report here the cloning and functional expression of a human ecto-ATPase. The study of the biochemical properties and the regulatory mechanisms of ecto-ATPases of defined sequence will be valuable in the definition of their role in nucleotide signalling.

| S-EPMC1571647 | biostudies-literature

Cloning of a polycistronic cDNA from tomato encoding gamma-glutamyl kinase and gamma-glutamyl phosphate reductase.

Project description:We isolated from a tomato cDNA library the tomPRO1 locus, which encodes gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E. coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.

| S-EPMC21589 | biostudies-literature

Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes.

Project description:We have cloned and sequenced a cDNA for a metacyclic trypomastigote-specific glycoprotein with a molecular mass of 90 kDa, termed MTS-gp90. By immunoblotting, antibodies to the MTS-gp90 recombinant protein reacted exclusively with a 90-kDa antigen of metacyclic trypomastigotes. The insert of the MTS-gp90 cDNA clone strongly hybridized with a single 3.0-kb mRNA of metacyclic forms, whereas the hybridization signal with epimastigote mRNA was weak and those with RNAs from other developmental stages were negative, indicating that transcription of the MTS-gp90 gene is developmentally regulated. A series of experiments showed that the MTS-gp90 gene is present in multiple copies in the Trypanosoma cruzi genome, arranged in a nontandem manner, and that there are at least 40 copies of the gene per haploid genome. Sequence analysis of recombinant MTS-gp90 revealed 40 to 60% identity at the amino acid level with members of a family of mammalian stage-specific, 85-kDa surface antigens of T. cruzi. However, there are considerable differences in the amino acid compositions outside the homology region.

| S-EPMC281144 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data