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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

APEX nuclease (Apex gene product) is a mammalian multifunctional DNA repair enzyme possibly involved in the repair of apurinic/apyrimidinic (AP) sites and single-strand DNA breaks with 3' termini blocked by nucleotide fragments and also in transcriptional regulation via redox activation of the AP-1 transcription factors. We cloned a 15-kb DNA fragment containing the Apex gene from a mouse leukocyte genomic library and determined a 4-kb stretch of its nucleotide sequence, including the complete sequence of the mouse Apex gene. The gene consists of 5 exons and 4 introns spanning 2.21 kb, and the boundaries between exons and introns follow the GT/AG rule. Two major and one minor transcription initiation sites were assigned to positions +1 and +24 and position +14, respectively, by a combination of ribonuclease protection, primer extension, and 5' RACE analyses. Position +1 is located 312 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exon II and exon V, respectively. The sequenced 5' flanking region (1.32 kb) lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors, such as ATF, NF-IL6, Sp1, and AP2. The 0.8-kb region from position -410 (5' flanking region) to position +386 (intron II) contains a CpG island. The Apex gene locus was mapped to mouse chromosome 14C2-D1 using in situ hybridization.
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PMID:Cloning, sequence analysis, and chromosomal assignment of the mouse Apex gene. 778 87

The Duffy gene has been shown not to be split by introns, even in its 5' untranslated region, and to be expressed not only in erythroid but in postcapillary venule endothelium of almost every organ in the body. To further investigate the transcriptional start position in erythroid and postcapillary venule endothelium, we performed 5'-rapid amplification of cDNA ends (5'-RACE). While every positive clone of 5'-RACE encoded the identical sequence of previously identified cDNA downstream from nucleotide 203, the upstream sequences were different. The upstream sequences corresponded to the sequence from nucleotide -279 to -308/-357 in erythroblasts and from -279 to -355/-383 in lung and were regarded as comprising a novel exon. This novel exon encoded seven residues initiated with a methionine, linked to nucleotide 203 in-frame and in agreement with the GT-AG splicing rule. The major erythroid transcriptional start position was identified in human erythroleukemia cells by primer extension and in bone marrow by ribonuclease protection analysis at 34 bases upstream from the first ATG codon. Distinctively, in lung and kidney, the transcription was started at 82 bases upstream from the ATG. Both Northern blotting and reverse transcription-polymerase chain reaction followed by Southern analysis indicated a predominance of the novel spliced form of mRNA of about 50- to 200-fold comparing with the unspliced form, in every studied organ and erythroid lineage cells. The spliced form of cDNA has been transfected into a human erythroleukemic cell line, K562, and the expressed protein reacted with Duffy-specific murine monoclonal antibody Fy6. These studies indicate that the product from the spliced form of mRNA is the major product of the Duffy gene in the erythroid lineage and postcapillary venule endothelium.
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PMID:Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium. 854 65

Diazepam-binding inhibitor (DBI)/acyl-CoA-binding protein (ACBP) is a highly conserved 10-kD polypeptide expressed in various organs and implicated in the regulation of multiple biological processes such as GABAA/benzodiazepine receptor modulation, acyl-CoA metabolism, steroidogenesis, and insulin secretion. To extend our knowledge about the biology of DBI/ACBP and to elucidate the molecular mechanisms responsible for regulating DBI/ACBP gene expression, we have studied the androgen-regulated expression of DBI/ACBP transcripts in the human prostatic adenocarcinoma cell line LNCaP and have cloned and characterized a human gene encoding DBI/ACBP. Northern blotting, reverse transcription-assisted polymerase chain reaction (RT-PCR), ribonuclease protection, and 5' RACE analysis (rapid amplification of cDNA ends) of DBI/ACBP transcripts in LNCaP cells revealed androgen-regulated expression of multiple transcripts originating from multiple transcription start sites and alternative processing. The most abundant type of transcripts (referred to as type 1 transcripts) encodes genuine DBI/ACBP of 86 amino acids, while the minor type (type 2 transcripts) harbors an insertion of 86 bases and might encode an unrelated protein of 67 amino acids. Examination of a cloned DBI/ACBP gene revealed a structural organization of four exons present in all transcripts and one alternatively used exon present only in type 2 transcripts. The promoter region is located in a CpG island and lacks a canonical TATA box. Transient transfection of DBI/ACBP promoter fragments into LNCaP cells demonstrated that a region of 1.1 kb upstream of the translation start site is able to drive high-level expression of luciferase in LNCaP cells in an androgen-regulated fashion. Taken together these data indicate that the isolated human gene encoding DBI/ACBP is functional, has a high degree of structural similarity with the corresponding rat gene, exhibits hallmarks of a typical housekeeping gene, and harbors cis-acting elements that are at least partially responsible for androgen-regulated transcription in LNCaP cells.
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PMID:A human gene encoding diazepam-binding inhibitor/acy1-CoA-binding protein: transcription and hormonal regulation in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP. 863 49

The recent cloning of several cDNAs encoding prostaglandin (PG) receptors has paved the way for a more detailed investigation of the postulated regulatory role of prostaglandins in corpus luteum function. We have utilized the reverse transcription-polymerase chain reaction (RT-PCR) to isolate a mRNA encoding the ovarian PGF(2alpha) (FP) receptor, using oligonucleotides based on the recently cloned mouse cDNA as primers. The 5'-untranslated region of the rat ovarian mRNA was isolated following 5'-RACE (rapid amplification of cDNA ends). The isolated 1526 base-pair sequence, which spans the entire open reading frame, was found 100% identical in the protein coding region to a similar sequence isolated from a rat astrocyte cDNA library, but different in the first 32 nucleotides of the 5'-untranslated region, possibly due to tissue-specific splicing heterogeneity. Using ribonuclease protection assay, a quantitative analysis of FP receptor mRNA levels was performed in corpora lutea excised from adult pseudopregnant rats (Day 8) at different timepoints (0.5-48 h) following the in vivo s.c. regimen of a luteolytic dose of the FP receptor agonist cloprostenol (5 microg). Already 3 h after cloprostenol injection, FP receptor mRNA levels exhibited a pronounced increase to values 4.0-fold higher (P < 0.01) than before injection. At 7 h through 24 h, the amount of luteal FP receptor mRNA decreased, approaching preinjection levels, whereafter they were again 3.0-fold higher (P < 0.01) at 48 h than before injection. We conclude that following homologous stimulation of the FP receptor, abundance of this mRNA is tissue-specifically regulated in a dynamic pattern, suggestive of an important role for FP receptor-mediated action on gene expression during the demise of corpus luteum function.
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PMID:Characterization and regulation of a mRNA encoding the prostaglandin F2alpha receptor in the rat ovary. 891 10

A genomic clone containing sequence identical to the 5' region of the cDNA for rat Type I hexokinase was isolated from a lambda Charon 4A library. A 5.4-kb EcoRI fragment from this clone, containing the matching sequence, was sequenced in its entirety. Rapid amplification of 5' cDNA ends (5' RACE), reverse transcription polymerase chain reaction, and ribonuclease protection experiments were consistent with the existence of multiple transcriptional start sites clustered in three regions approximately 460, 300, and 100 nucleotides upstream from the translational start codon. Together with results of previous work, the 5' untranslated sequence defined in the present study accounts for the 4.3-kb mRNA for Type I hexokinase seen on Northern blots. Fragments from the 5' flanking region were cloned into a reporter vector containing the luciferase coding region. Based on transfection experiments with both PC12 and H9c2 cells, promoter activity was associated with a region lying between nucleotide positions -742 and -516 (with A of the ATG codon at the translational start site defined as +1). The promoter region lacks a TATA sequence and, together with the transcriptional start sites, is located within a GC rich segment (a "CpG island") approximately 1 kb in length. These characteristics have previously been associated with the promoter and transcriptional start sites of genes for "housekeeping enzymes."
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PMID:Isolation of the promoter for Type I hexokinase from rat. 891 47

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5'- and 3'-RACE technique. The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.
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PMID:The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease. Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity. 949 70

Three distinct transforming growth factor-beta1 (TGF-beta1) transcripts of 2.5, 1.9 and 1.4kb have been described, but the start sites and functional significance of the shorter transcripts are unknown. Here, we have cloned and sequenced a rat genomic fragment encoding approximately 1250 bases upstream of the start of the TGF-beta1 open reading frame. Using a combination of ribonuclease protection and 5' RACE-PCR analysis, we have mapped the start sites for the two shorter TGF-beta1 transcripts in NRP152 cells, a rat prostatic epithelial cell line that expresses all three transcripts at high levels. The 1.4-kb mRNA starts 25 bases upstream of the initiator AUG, whereas the 1.9-kb mRNA has two start sites 366 and 401 bases upstream of the AUG. Polysome analysis of the NRP152 cells indicates that the 1.9-kb transcript is very efficiently translated, whereas the 2.5- and 1.4-kb transcripts appear to be poorly translated. Differential regulation of TGF-beta1 transcript size may therefore represent an important mechanism for regulating TGF-beta1 protein levels.
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PMID:Identification of the start sites for the 1.9- and 1.4-kb rat transforming growth factor-beta1 transcripts and their effect on translational efficiency. 975 3

Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily.
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PMID:Isolation and sequencing of a cDNA encoding for a ribonuclease from the insect Ceratitis capitata. 1069 91

Hypothalamic sensitivity to leptin has been suggested to be important for regulation of body fat mass. Mice heterozygous for a mutation in the leptin receptor (leptin-R) have an increased body fat mass suggesting that the abundance of leptin-R may be an important determinator of leptin sensitivity. Leptin-R cDNAs from several species contain alternative 5'untranslated regions (5'UTRs), suggesting that several distinct regulatory regions may exist. To investigate possible mechanisms by which leptin-R expression may be regulated, we searched for possible alternative 5'UTRs of the leptin-R in the rat and determined their location in relation to putative response elements. Four leptin-R 5'UTRs (exons 1A-1D), which diverged 23 bp upstream of the start codon, were identified by 5'Rapid Amplification of cDNA Ends (5'RACE) and sequencing. Exons 1B and 1C were present in 31 and 61%, respectively, of all leptin-R transcripts in the hypothalamus as determined by a ribonuclease protection assay. Analysis of the 5' flanking genomic sequences revealed an imperfect estrogen response element (ERE), two Spl-sites, three CCAAT-boxes and one octamer. Exons 1A and 1D corresponded to a putative second gene, encoding the OB-Receptor Gene Related Protein (OB-RGRP), which is transcribed from a promoter shared with the leptin-R. DNA sequencing revealed that the rat OB-RGRP had 98 and 97% homology with the mouse and human sequence, respectively. We report here that transcription of the rat leptin-R gene may generate transcripts with four alternative 5'UTRs. The presence of a putative ERE, close to the most frequently used transcriptional start sites of the leptin-R gene in the hypothalamus, provides a possible mechanism by which estrogen may exert its effects on food intake.
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PMID:Leptin receptor 5'untranslated regions in the rat: relative abundance, genomic organization and relation to putative response elements. 1116 38

The structure of isotype-specific regions of classes 1, II, III, IVa and IVb of canine beta-tubulin was characterized by 3'-RACE and the expression of these isotypes in canine tissues was examined by ribonuclease protection assay (RPA). Furthermore, a malignant mammary tumor-derived osteosarcoma-like cell line was established and the altered expression of beta-tubulin isotypes in taxol-resistant sublines was analyzed. The deduced amino acid sequences in isotype-specific regions corresponding to classes I, II and IVb were identical to those of humans and mice, but those in classes III and IVa showed slight differences among species. RPA revealed that classes I and IVb were widely distributed, but classes II, III and IVa were restricted to the brain. Because RPA could clearly distinguish the expression of class IVa from that of class IVb, it was thought to be more useful than northern blot for analysis of beta-tubulin isotype expression. In vitro, taxol-resistant sublines displayed a significant increase in class IVa as compared with taxol-sensitive cells, suggesting that altered expression of class IVa was associated with taxol resistance in these cell lines.
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PMID:Characterization of isotype-specific regions of five classes of canine beta-tubulin and their expression in several tissues and cell culture. 1178 7


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