Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3'-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5' end of the cDNA was amplified by a 5'-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.
Plant Mol Biol 1994 Oct
PMID:Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase. 794 18

A second gene (rp-L21) copy, clone g34, coding for ribosomal (r-) protein L21, was isolated from the pathogenic (P) strain HM-1:IMSS cl6 of the intestinal parasite Entamoeba histolytica (Eh). The gene was compared to the previously isolated copy, gLE3 [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], with respect to its primary structure, mRNA levels and binding to the r-complex during translation. Unlike the gLE3 gene copy [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], g34 was found not to be physically connected to an actin gene copy. Homologous copies of the two rp-L21 genes were also characterized from the nonpathogenic (NP) strain SAW1734R clAR, as well as from its P derivative. Sequence comparison of the coding regions of the two rp-L21 revealed almost full identity. Significant differences were found, however, within their 3' and 5' flanking regions. Using the 3' rapid amplification of cDNA ends (3' RACE) method [Frohman et al., Proc. Natl. Acad. Sci. USA 85 (1988) 8998-9002], as well as Northern and slot blot hybridizations, it was demonstrated that both rp-L21 mRNAs are found in similar amounts. However, as was shown by differential hybridization, the relative binding of each transcript to the r-complex varied somewhat between P and NP strains. This finding suggests that the control of expression of rp-L21 in Eh may involve regulation at the post-transcriptional level.
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PMID:Characterization of two distinct gene transcripts for ribosomal protein L21 from pathogenic and nonpathogenic strains of Entamoeba histolytica. 795 49

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamine/glutamic acid-rich region encoded by the C-terminal part of the alpha chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342-350) and limited own data.
Plant Mol Biol 1994 Apr
PMID:The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes. 800 94

Estrogens are synthesized from C19 steroids by a unique form of cytochrome P450, aromatase cytochrome P-450 (P-450AROM; the product of the CYP19 gene). We have shown that tissue-specific expression of human P-450AROM is determined, in part, by the use of alternative promoters. Previous methods of analysis for determining the specific 5'-termini of the different transcripts included S1 nuclease protection, primer extension, and Northern analysis. In the present study we have used the RACE procedure (rapid amplification of cDNA ends) to amplify and clone the 5' termini of P-450AROM transcripts expressed in human corpus luteum (CL). Sequencing of the resulting clones supports the results of the previously performed studies. Specifically, the proximal promoter, PII, is the predominant promoter utilized in CL, such that the start of transcription occurs 26 bp downstream of the putative TATA sequence. A minority of the clones possess an alternative 5'-end, namely I.3. Exon-specific Northern analysis confirms that the majority of the P-450AROM transcripts in CL tissue contain sequence specific for promoter II. Similarly, exon-specific Northern analysis indicates that transcripts in human follicles, as well as granulosa cells in culture, contain primarily sequence specific for promoter II.
Mol Cell Endocrinol 1993 Nov
PMID:Exon-specific northern analysis and rapid amplification of cDNA ends (RACE) reveal that the proximal promoter II (PII) is responsible for aromatase cytochrome P450 (CYP19) expression in human ovary. 814 90

Surprisingly half of all severe haemophilia A patients have no mutation in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect that prevents the amplification of the message across the boundary between exon 22 and 23. This locates the defect to internal regions of intron 22. Novel sequences 3' to exon 22 were isolated from the 9 available patients with the above abnormality by combining RACE and vectorette amplifications on trace amounts of mRNA. This showed that exons 1-22 of the factor VIII mRNA had become part of a hybrid message containing new multi exonic sequences expressed in normal cells. The novel sequences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones covering intron 22 showed no obvious abnormalities. This suggested inversions involving intron 22 repeated sequences. Screening of 3 YAC libraries with the novel sequences located them at least 200 kb telomeric (5') to factor VIII and pulsed field gel analysis detected abnormal bands in patients. This demonstrates that the mutations in the patients are inversions of long DNA regions possibly involving the repeated sequences and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.
Hum Mol Genet 1993 Nov
PMID:Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions. 828 Nov 36

The human MIC2 gene is pseudoautosomal and in females it escapes X inactivation. At the 5' end of the gene a 1.2-kb-long CpG island has been identified that is unmethylated on the active X, the inactive X, and on the Y chromosome. We have demonstrated by 5' RACE experiments that this region contains the transcription start site of the gene. To better characterize this CpG island, we have investigated the interaction between this region and nuclear proteins in vitro by using DNA gel mobility shift and DNase I footprinting techniques. Band shift experiments with HeLa cell nuclear extract have indicated that all the island is involved in multiple interactions with nuclear proteins. Experiments with a eukaryotic purified Sp1 protein have shown that this factor specifically binds to several sites of the island. Three DNase I protected footprints have been identified in the region between nucleotides -122 and +34 with respect to the transcription initiation site. By using a recombinant Sp1 protein, we have shown that all the footprints are due to the binding of Sp1. The sequences of two footprints correspond to the decanucleotide binding site for Sp1, the sequence of the third one does not contain any published Sp1 recognition site.
Somat Cell Mol Genet 1993 Jan
PMID:Multiple DNA-protein interactions at the CpG island of the human pseudoautosomal gene MIC2. 846 Mar 98

The complete sequence of the cDNA encoding the nematode polyprotein allergen/antigen (NPA) of the bovine lungworm Dictyocaulus viviparus was obtained by immunoscreening of cDNA expression libraries and by 5' RACE (rapid amplification of cDNA ends). The encoded polypeptide is similar in sequence to the ABA-1 allergen of Ascaris, the gp15/400 'ladder' protein of Brugia malayi, Brugia pahangi and Wuchereria bancrofti, and a 15-kDa antigen of Dirofilaria immitis. As with these, the predicted amino-acid sequence comprises a head-to-tail array of similar polypeptides with regularly spaced consensus proteinase cleavage sites. The D. viviparus protein was designated DvA-1 (D. viviparus antigen-1) and the gene dva-1. The deduced amino-acid sequence of DvA-1 showed features not observed before in other NPAs: (i) a hydrophobic leader peptide is present, (ii) none of the 12 units in the array are identical and the sequences diverge to a degree hitherto unseen in the NPAs of other nematode parasites, (iii) the predicted proteinase cleavage sites are also diverse in sequence and, in two instances, no consensus cleavage site was identifiable at the expected position, (iv) a short repeat unit is present, which is the only one containing a consensus N-glycosylation site and (v) a C-terminal extension peptide is encoded which shows no similarity to that from A. suum ABA-1. Comparison of independent cDNAs revealed slight variations in the sequence of the gene within the parasite population. Antisera to recombinant DvA-1 polypeptide identified 14-15-kDa antigens in both parasite somatic and excretory-secretory material. DvA-1 is the only NPA for which the complete coding sequence is available and the new principles which it illustrates may lie unsuspected in the NPA-encoding genes of all nematode parasites.
Mol Biochem Parasitol 1995 Jun
PMID:Extensive diversity in repeat unit sequences of the cDNA encoding the polyprotein antigen/allergen from the bovine lungworm Dictyocaulus viviparus. 853 2

By screening a human fetal brain cDNA expression library using a monoclonal antiphosphotyrosine antibody and by 5' RACE procedures, we have isolated overlapping cDNAs encoding a receptor-type tyrosine kinase belonging to the EPH family, DRT (Developmentally Regulated EPH-related Tyrosine kinase gene). The DRT gene is expressed in three different size transcripts (i.e. 4, 5 and 11 kb). DRT transcripts are expressed in human brain and several other tissues, including heart, lung, kidney, placenta, pancreas, liver and skeletal muscle, but the 11 kb DRT transcript is preferentially expressed in fetal brain. Steady-state levels of DRT mRNA in several tissues, including brain, heart, lung and kidney, are greater in the midterm fetus than those in the adult. DRT transcripts are detectable at low levels in a human teratocarcinoma cell line (NTera-2), but its expression is greatly increased after the NTera-2 cells are induced to become postmitotic neurons (NTera-2N) by retinoic acid treatment. These data suggest that DRT plays a part in human neurogenesis. A large number of tumor cell lines derived from neuroectoderm express DRT transcripts, including 12 neuroblastomas, two medulloblastomas, one primitive neuroectodermal tumor and six small cell lung carcinomas (SCLC). Interestingly, several neuroblastoma cell lines with 1p deletion and one SCLC cell line express DRT transcripts of aberrant size (i.e. 3, 6 and 8 kb) in addition to those found in normal tissues. We mapped the DRT gene to human chromosome 1p35-1p36.1 by PCR screening of human-rodent somatic cell hybrid panels and by fluorescence in situ hybridization. As the distal end of chromosome 1p is often deleted in neuroblastomas and altered in some cases in SCLCs, these chromosomal abnormalities may have resulted in the generation of aberrant size transcripts. Thus, the DRT gene may play a part in neuroblastoma and SCLC tumorigenesis.
Hum Mol Genet 1995 Nov
PMID:Molecular characterization and chromosomal localization of DRT (EPHT3): a developmentally regulated human protein-tyrosine kinase gene of the EPH family. 858 79

Complement component C4 is encoded by two nearly identical genes, C4A and C4B, that encode a C4 precursor that is proteolytically cleaved into the alpha, beta and gamma subunits of the mature protein. C4 is expressed primarily in liver and to a much lesser extent in immune cells. We have identified a unique 1 kb RNA transcript, termed Z, that arises from a cryptic promoter lying in the intron between exons 35 and 36 of the C4 gene. Primer extension, RNase protection, and 5' RACE experiments locate the cap site in intron 35, 55 bases upstream from exon 36. Northern blotting and RNase protection assays show that expression of this 1 kb Z RNA transcript is confined to the adrenal gland. Z RNA contains the same open reading frame as C4 which predicts a protein of 131 amino acids, but antisera to C4 do not interact with epitopes on this protein when it is synthesized by cell-free translation, hence the presence or absence of a Z protein in vivo could not be determined. Transfection of Z promoter/reporter constructs into human adrenal NCI-H295 cells shows that most if not all of the sequences required for high-level adrenal expression lie within 235 bases upstream from the cap site, but that this region is inactive when transfected into COS-1, JEG-3 and Hep-G2 cells, suggesting it contains an adrenal-specific element. The 222 bases upstream from the cap site are 75% identical in the human C4A and mouse Slp genes, and contain a potential binding site for steroidogenic factor 1 (SF-1), an orphan zinc-finger nuclear receptor. We propose that this region, like a nearby region in the mouse genome, functions as an upstream element of the P450c21 promoter, and may be a component of an adrenal-specific locus-control region.
Hum Mol Genet 1995 Nov
PMID:A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element? 858 88

A previous study has demonstrated that the carboxyl-terminal (C-terminal) processing protease in spinach for the D1 precursor protein (pDl) of the photosystem II reaction center is a monomeric protein of about 45 kDa. Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme has been identified and sequenced, from a spinach green leaf cDNA library. In order to determine the 5' end of the transcript, the rapid amplification of cDNA end (5'-RACE) technique was applied. By these analyses, the full-length transcript was established to consist of 1906 nucleotides and a poly(A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra amino-terminal extension characteristic of both a transit peptide and a signal sequence. This suggests that the protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids. The mature part of the enzyme consists of 389 amino acid residues and exhibits a significant sequence homology with two groups of proteins as demonstrated by a computer homology search, i.e. (1) the deduced sequence of a protein proposed to be the C-terminal processing protease for pD1 in Synechocystis sp. PCC 6803, based on genetic experiments and (2) proteases for C-terminal cleavage identified in Escherichia coli and Bartonella bacilliformis.
Plant Mol Biol 1996 Jan
PMID:Carboxyl-terminal processing protease for the D1 precursor protein: cloning and sequencing of the spinach cDNA. 861 42


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