Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a novel point mutation in the fourth exon of human factor IX (encoding the first EGF-like domain) in which cytosine is substituted for adenosine at position 10,401, resulting in the substitution of proline for glutamine at position 50 in the polypeptide chain. Sequence analysis of all eight exons, all exon-intron junctions, 160 base pairs (bp) of DNA 5' to the proposed translation start site, and 60 bp 3' to the translation termination site shows no other difference from the normal factor IX gene, with the exception of a previously described benign polymorphism at position 148 in the protein (Ala----Thr). The affected subject has severe hemophilia B with no detectable factor IX activity despite normal factor IX antigen levels. We purified the abnormal factor IX by immunoaffinity chromatography and demonstrated that its activation by factor Xla is markedly delayed compared with normal factor lX. Once activated, the abnormal factor lX binds antithrombin III in a 1:1 molar ratio, and the activated protein demonstrates catalytic activity, suggesting an intact active site. The mutation creates a new Bst Yl restriction endonuclease cleavage site. Restriction with Bst Yl shows the mutation in maternal DNA and offers the possibility of direct carrier status analysis and prenatal diagnosis in kindreds with this mutation. We designate this new mutation factor lXNew London. This is the only reported mutation in the first EGF-like domain that causes severe hemophilia B.
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PMID:Factor IX New London: substitution of proline for glutamine at position 50 causes severe hemophilia B. 230 16

Four distinct intragenic polymorphisms in the coagulation factor IX gene which have been reported to be important for family diagnosis of Caucasian hemophilia B were studied in 51 normal Japanese subjects (21 males and 30 females). High-molecular-weight DNA prepared from peripheral blood lymphocytes were digested with endonuclease, Ddel, Mspl, Taql or Xmnl, and were studied by Southern blot analysis with factor IX complementary DNA as a probe. None of the minor fragments produced by these enzymes was found in the normal Japanese DNA samples tested, although the probe detects minor allelic forms in control Caucasian DNA samples. Our data suggest that the frequent polymorphic sites found in Caucasians are possibly absent in the Japanese population.
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PMID:Possible absence of common polymorphisms in coagulation factor IX gene in Japanese subjects. 287 95

Hemophilia B or Christmas disease is an X-linked condition caused by absent or reduced levels of functional coagulation factor IX. Based upon the peptide sequence of bovine factor IX, we synthesized a 17-base pair oligonucleotide probe to screen a human liver cDNA library. A recombinant clone was identified with a 917-nucleotide insert whose sequence corresponds to 70% of the coding region of human factor IX. This factor IX cDNA was used to probe restriction endonuclease digested human DNA to identify a Taq I polymorphism associated with the genomic factor IX gene as well as to verify that there is a single copy of this gene per haploid genome. The factor IX cDNA was also used to map the locus for factor IX to a region from Xq26 to Xqter. The cloning of human factor IX cDNA and identification of a Taq I polymorphism and its regional localization will provide a means to study the molecular genetics of hemophilia B and permit linkage analysis with nearby loci.
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PMID:Isolation and characterization of human factor IX cDNA: identification of Taq I polymorphism and regional assignment. 608 57

Hemophilia B Leyden is a rare form of congenital factor IX deficiency which is characterized by severe factor IX deficiency at birth, which ameliorates after puberty. It is caused by mutations in the factor IX gene promoter region and the postpubertal amelioration is thought to be mediated by the action of testosterone on an androgen response element located in the promoter region. Three kindreds have been previously reported with a milder form of hemophilia B Leyden, associated with a guanine to adenine transition at nucleotide position -6 of the promoter region. We now report a fourth kindred with this mutation. The proband was a newborn with a factor IX level of 2.5%, his 12-year-old half-brother had a level of 28%, and his mother's 56-year-old maternal cousin had a level of 60%. A G to A transition at nucleotide -6 of the promoter region was demonstrated by cloning and sequencing polymerase chain reaction products from the half brother, and the mother was demonstrated to be a carrier. The mutation eliminates a TaqI restriction endonuclease site normally present in the wild type promoter, and the mother's cousin was demonstrated to carry the mutation by the absence of digestion with TaqI. The identification of hemophilia B Leyden with this specific mutation has practical importance to the clinical management because of its unique natural history and significantly better prognosis than classical hemophilia B.
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PMID:Moderate hemophilia B Leyden: identification by polymerase chain reaction, sequencing, and oligomer restriction. 760 25

Restriction endonuclease fingerprinting (REF) is a modification of single-strand confirmation polymorphism (SSCP) that was developed to detect the presence of essentially all mutations in a 1-kb segment. To test REF, a 1-kb segment of the human factor IX gene was amplified with PCR and digested with each of five groups of restriction endonucleases. The endonucleases in each group were chosen so that the average size of the fragments was about 150 bp. After separate digestions, the products were mixed, 5' end-labeled with T4 polynucleotide kinase, denatured and electrophoresed under nondenaturing conditions. Each lane screened 1 kb and typically contained 68 segments (6.8 fragments per average digestion x 5 digestions x 2 strands). REF was performed with 5.6% polyacrylamide and 7.5% GeneAmp at temperatures of either 23 degrees or 8 degrees C. Point mutations resulted in the gain or loss of a restriction site in 21% of 24 test mutations (informative restriction component). In cases in which the restriction component was not informative, mutations were detected if any of the five mutation-containing restriction fragments (producing 10 single-stranded segments) displayed abnormal mobility (SSCP component). The average efficiency per single-stranded segment of the SSCP component for the 24 point mutations ranged from 49% for polyacrylamide at 23 degrees C to 68% with GeneAmp at 8 degrees C. REF detected 96% of the mutations with polyacrylamide at 23 degrees C and 100% with GeneAmp at 23 degrees or 8 degrees C. GeneAmp at 23 degrees and 8 degrees C also detected 100% of a subsequent blinded sample that contained normal controls and 27 different point mutations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Restriction endonuclease fingerprinting (REF): a sensitive method for screening mutations in long, contiguous segments of DNA. 777 98

New systems are proposed for the PCR analysis of HindIII polymorphic sites in the gamma A and gamma G globin genes and of TaqI polymorphic site in the human factor IX gene of blood population. DNA fragments amplified according to the systems described contain constant restriction site of the appropriate endonuclease, in addition to the polymorphic one, which significantly improves the reliability of the RELP analysis. The systems proposed are highly specific and may be used for DNA diagnosis of beta-thalassemia and haemophilia B.
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PMID:[A PCR-system of analyzing polymorphic markers in gamma-A and gamma-G globin genes and gene for human blood coagulation factor IX with an internal control of the completeness of restriction hydrolysis]. 809 4

The authors are reporting on the prenatal diagnosis of the X-linked haemophilia B for the first time in Hungary applying the polymerase chain reaction. DNA sequence containing a HhaI restriction endonuclease site close to the factor IX gene was amplified using polymerase chain reaction. The products from polymerase chain reaction were detected on polyacrylamide gel with ethidium bromide staining after the digestion with HhaI restriction enzyme. In the first step of the diagnosis DNA specimen was prepared from chorion derived from a 11th week gestation of haemophilia B carrier mother. The investigation of fetal DNA proved a male fetus. The detection of HhaI polymorphism of the fetus demonstrated the inheritance of the disease causing allele. The parents asked for the termination of pregnancy based on the result.
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PMID:[Prenatal diagnosis of hemophilia B]. 918 75

We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage oC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The oC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications.
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PMID:Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. 1570 95

Persistence of transgene expression is a major limitation for nonvirus-mediated gene therapy approaches. We have suggested that covalent linkage of bacterial DNA to the expression cassette plays a critical role in transcriptional silencing of transgenes in vivo. To gain insight into the role of the covalent linkage of plasmid DNA to the expression cassette and transcriptional repression, and whether this silencing effect could be alleviated by altering the molecular structure of vector DNAs in vivo, we generated a scheme for converting routine plasmids into a purified expression cassette, free of bacterial DNA after gene transfer in vivo. To do this, the human alpha-1-antitrypsin (hAAT) and human clotting factor IX (hfIX) reporter genes were flanked by two ISceI endonuclease recognition sites, and coinjected together with a plasmid encoding the I-SceI cDNA or a control plasmid into mouse liver. Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed low serum levels of hAAT or hFIX, which remained low throughout the length of the experiment. However, animals that expressed I-SceI had a 5- to 10-fold increase in serum hAAT or hFIX that persisted for at least 8 months (length of study). Expression of I-SceI resulted in cleavage and excision of the expression cassettes from the plasmid backbone, forming mostly circles devoid of bacterial DNA sequences, as established by a battery of different Southern blot and polymerase chain reaction analyses in both C57BL/6 and scid treated mice. In contrast, only the input parental circular plasmid DNA band was detected in mice injected with the reporter gene alone, or an I-SceI plasmid together with the hAAT reporter plasmid lacking the I-SceI sites. Similar results were obtained when the Flp recombinase system was used to make mini-plasmids in mouse liver in vivo. This study presents further independent evidence that removing the covalent linkage between plasmid and transgene sequences leads to a marked increase in and persistence of transgene expression. Unraveling the mechanisms by which the covalent linkage of bacterial DNA to the expression cassette is connected to gene silencing is fundamental to establishing the mechanism of transcriptional regulation in mammalian systems and will be important for the development of versatile nonviral vectors that can be used to achieve persistent gene expression in different cell types.
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PMID:Increased maintenance and persistence of transgenes by excision of expression cassettes from plasmid sequences in vivo. 1591 81