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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene for rabbit alpha 1 acid glycoprotein (AGP) has been isolated from a lambda EMBL3 genomic DNA library. Isolated clone contains a 12 Kbp fragment of rabbit genomic DNA. Restriction endonuclease mapping has localized the gene within a 4.2 Kbp fragment spanning two EcoRI sites. Southern blot analysis of the rabbit genomic DNA and its comparison with the cloned gene indicates that there is only one gene for AGP present per genome. DNA sequence analysis of the cloned gene indicates that the entire gene, TATA box to the polyadenylation signal, is located within the 4.2 Kbp region and contains six exons representing the full-length cDNA described earlier (1). The 5'-end of alpha 1-AGP gene sequences from rabbit, human, rat and mouse have been compared. Such analysis reveals two conserved regions located between -63 bp and -36 bp and -29 bp and -1 bp of putative transcription start site, which may play a role in transcriptional induction of this gene during acute response. In addition to this conserved domain, DNA sequence upstream of the major transcription start site contains a potential element for Sp1 binding and a 18 bp long palindrome sequence followed by a short repeating dinucleotide sequence, which may be important in the regulation of AGP gene induction.
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PMID:Cloning and structural characterization of a rabbit genomic DNA for alpha 1 acid glycoprotein. 153 58

Previous studies have demonstrated that the mouse c-Harvey ras proto-oncogene (c-Ha-ras) promoter sequences are GC rich and contain several potential transcription factor SP1 binding sites. We investigated the endonuclease hypersensitivity of this region in nuclei in vitro and whole mouse tissues in vivo and identified a very strong, ubiquitous hypersensitive site covering the proximal promoter sequences. Footprint protection studies using nuclear extracts from various cell types including fibroblasts, erythroid cells, and both normal and transformed epithelial cells revealed a consistent protein-binding pattern. Five protein binding sites were observed, four of which correlated with potential SP1 binding sites. Competition experiments using an oligonucleotide corresponding to a consensus SP1 binding site confirmed that these sequences were indeed bound by the SP1 (or SP1-like) trans-acting factor. In addition, no differences were observed between the footprint patterns obtained using extracts from cells of different lineages or between normal and transformed epithelial cells carrying activated ras genes. The controlling elements responsible for differential c-Ha-ras transcription between cell types or at different stages of carcinogenesis therefore probably lie in other regions of the gene.
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PMID:Structural analysis of the mouse c-Ha-ras gene promoter. 204 51

In a previous report, we localized the gene for a 130-kilodalton envelope glycoprotein (gI) of bovine herpesvirus 1 (BHV-1) to a 3.6-kilobase HpaI-KpnI restriction endonuclease fragment from the long unique region of the BHV-1 genome (map position 0.405 to 0.432) and showed that a herpes simplex virus 1 (HSV-1) glycoprotein B (gB) probe uniquely hybridized to this BHV-1 restriction fragment. Here we present the complete nucleotide sequence of the BHV-1 gI gene and the predicted 932-amino-acid sequence of the gI primary translation product. Comparison with the published nucleotide sequence of the HSV-1 (KOS) gB gene (D. J. Bzik, B. A. Fox, N. A. DeLuca, and S. Person, Virology 133:301-314, 1984) reveals a similarity of 56.3% at the nucleotide level and 45.9% at the amino acid level. Upstream of the proposed gI coding region are potential mRNA transcriptional promoter elements including a TATA box and multiple Sp1 binding sites (GC boxes). Downstream of the gI coding region are two sequence elements associated with mRNA cleavage and polyadenylation (AATAAA and a GT-rich region roughly 30 nucleotides further downstream). Like HSV-1 gB, the predicted gI amino acid sequence exhibits two broad hydrophobic regions likely to represent a transient amino-terminal signal sequence and a transmembrane anchor domain (near the carboxyl terminus). Additional features shared with gB include 6 potential N-linked glycosylation sites and 10 highly conserved cysteine residues in the gI extracellular domain. Two regions of nonsimilarity between gI and gB are a centrally located 22-amino-acid region of gI for which there is essentially no gB counterpart and the transient amino-terminal leaders which differ in both size and sequence. The hydrophobic signal sequence of the gI leader, unlike that of gB, is preceded by an unusually large region of predominantly hydrophilic amino acids. The unusual length of the gI leader may result from an overlap between that portion of the gI coding region and a potential upstream coding region.
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PMID:Comparison of the bovine herpesvirus 1 gI gene and the herpes simplex virus type 1 gB gene. 284 84

Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.
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PMID:Characterization of the promoter region of the human apurinic endonuclease gene (APE). 753 97

Transcobalamin II (TCII) is a plasma protein that binds vitamin B12 (cobalamin; Cbl) and facilitates the cellular uptake of the vitamin by receptor-mediated endocytosis. In genetic disorders that are characterized by congenital deficiency of TCII, intracellular Cbl deficiency occurs, resulting in an early onset of megaloblastic anemia that is sometimes accompanied by a neurologic disorder. To define the genetic basis for TCII deficiency, we have cloned and characterized the human gene that encodes this protein. The gene spans a minimum of 18 kbp and contains nine exons and eight introns, with a polyadenylation signal sequence located 509 bp downstream from the termination codon and a transcription initiation site beginning 158 bp upstream from the ATG translation start site. The 5' flanking DNA does not have a TATA or CCAAT regulatory element, but a 34-nucleotide stretch beginning just upstream of the CAP site contains four tandemly organized 5'-CCCC-3' tetramers. This sequence is a motif for a trans-active transcription factor (ETF) that regulates expression of the epidermal growth factor receptor gene (EGFR), which also lacks TATA and CCAAT regulatory elements. A GC-rich sequence that binds the SP1 protein is located 356 nucleotides upstream from the first of the series of CCCC tetramers. Although this GC sequence is at an unusual location with respect to the CAP site, a 507-bp fragment containing this GC box drives the chloramphenicol acetyltransferase (CAT) reporter gene after transient transfection into NIH 3T3 cells. No CAT activity was observed when a 420-bp fragment lacking this GC box but containing the ETF-binding domains was similarly transfected into this cell line. One consensus and two atypical motifs for the c-myc ligand are located downstream and upstream, respectively, of the GC box, and this could explain the elevated plasma TCII observed in some patients with multiple myeloma, as the c-myc product is overexpressed in some myeloma cells. Restriction endonuclease digestion of genomic DNA from eight normal subjects with Taq I, Hinfl, Msp I, and Bgl I identified three patterns of restriction fragment length polymorphism (RFLP). A number of the exon/intron splice junctions of human TCII, TCI, and IF genes are located in homologous regions of these proteins, providing evidence that these genes have evolved by duplication of an ancestral gene. This characterization of the TCII gene and the RFLP should facilitate the identification of the mutation(s) responsible for the genetic abnormalities of TCII expression.
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PMID:The cloning and characterization of the human transcobalamin II gene. 774 31

The structural organization of the gene for the E3 subunit of the human alpha-ketoacid dehydrogenase complexes, dihydrolipoamide dehydrogenase (DLD), and its upstream elements have been determined by restriction endonuclease mapping and DNA sequence analysis of overlapping genomic clones. The gene is approximately 20 kb long. It contains 14 exons ranging in size from 69 to 780 bp and 13 introns ranging in size from 93 bp to 7.0 kb. All splice donor and acceptor sites conform to the GT/AG rule. The 5' ends of mRNA transcripts upstream from the translation initiation codon were determined by primer extension assay. A "CAAT box"-like sequence is present at 39 bp upstream of the presumptive cap site and the 5' flanking region has been sequenced up to 2.0 kb upstream. There are several sequences compatible with presumptive promoter elements, including an Sp1 binding site, a nuclear respiratory factor 1 site, two cyclic AMP response element binding sites, and a possible negative response element present in the insulin promoter. A 313-bp segment from -2076 to -1763 is 89% homologous to a recently described pTR5 repetitive element found in the human genome.
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PMID:The structure of the human dihydrolipoamide dehydrogenase gene (DLD) and its upstream elements. 840 89

A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1. The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites. Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites. Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence. The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects. The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix.
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PMID:Splase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites. 889 94

We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and approximately 290 bp of the transcribed region linked to a chloramphenicol acetyltransferase (CAT) reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of approximately 190 bp of upstream and approximately 170 bp of transcribed DNA (exon 1 and most of intron 1). This approximately 360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5'-most CCAAT box decreased activity approximately 5-fold. The second CCAAT box (situated in exon 1) may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Spl site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like (USF) sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had approximately 5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells.
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PMID:Comparison of the promoters of the mouse (APEX) and human (APE) apurinic endonuclease genes. 950 86

CD38 is a leukocyte activation antigen and ectoenzyme [NAD(P)+ glycohydrolase; EC 3.2.2.6] involved in numerous immune functions. The human CD38 gene is complex [eight exons, >80 kilobases (kb) long] located on Chromosome 4p15, and part of the eukaryotic NAD+ glycohydrolase/ADP-ribosyl cyclase gene family. Because of the increasing relevance of the CD38 molecule in the host immune response to infectious, tumoral, and metabolic diseases, we investigated the genetic variability and linkage of the human CD38 locus. We report that (1) the restriction endonuclease Pvu II identifies a bi-allelic polymorphism here defined as formed by the alleles CD38*A (12 kb) and CD38*B (9/2.5 kb); (2) their frequency in the healthy Italian Caucasian population is 14% and 86%, respectively; (3) the polymorphic Pvu II site is located at the 5' end of the first intron of the CD38 gene; (4) in conjunction with the polymorphic site, we identified a 900 base pair CpG island associated with the CD38 gene, with two potential Sp1 binding sites; (5) the CpG island may play a role in the regulation of CD38 expression and is hypomethylated in various cell lines; (6) by pulsed-field gel electrophoresis we show that CD38 and its paralogue, the bone-marrow stromal cell antigen BST-1 (CD157), map to the same 800 kb Avi II fragment, indicating that the two human ecto-NADase genes are closely linked.
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PMID:The human CD38 gene: polymorphism, CpG island, and linkage to the CD157 (BST-1) gene. 1036 16

Prior studies demonstrated that expression of the retinoblastoma (RB) protein in acute myelogenous leukemia (AML) is heterogeneous with low expression conferring a poor prognosis. The molecular change(s) responsible for low RB expression in AML are unknown. Since methylation of the RB promoter has been shown to result in decreased expression we hypothesized that this might explain some cases of low RB expression in AML. To investigate this hypothesis Southern blotting and PCR sequencing after bisulfite conversion were used to study the methylation status of the RB gene promoter. DNA and protein lysates were prepared from the mononuclear cell fraction from peripheral blood or bone marrow samples from 46 patients with newly diagnosed AML. By Western blot 16, 22 and 8 patients had low, elevated and hyperphosphorylated patterns of RB expression respectively using previously defined criteria. The SacI endonuclease cuts a 5.7-kb or 6.8 -kb fragment, depending on polymorphism, containing the RB promoter, detected by the probe p123M1.8 that covers the RB promoter region and exon 1. The methylation sensitive endonuclease SacII cuts twice within a key hairpin loop structure in the RB promoter that contains binding sites for AP1, Sp1 and RBF1. Others have demonstrated that methylation within this hairpin loop can decrease RB mRNA transcription by up to 92%. Comparison of the SacI and SacI + SacII digestion fragments showed no evidence of methylation in the promoter region of RB in any of the patients studied. DNA from the promoter region of 11 patients with no/low RB expression was subjected to bisulfite conversion and PCR sequencing. No evidence of methylation was seen by this method either. These results suggests that hypermethylation of the RB promoter region is at best an infrequent event in AML and that RB promoter hypermethylation is not the predominant cause of the low levels of RB expression observed in 20% of AML patients.
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PMID:Altered expression of retinoblastoma (RB) protein in acute myelogenous leukemia does not result from methylation of the Rb promotor. 1070 51


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