Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNase I sensitivity of three different chromatin regions in mouse testicular cells was analysed by in situ nick translation with biotin-dUTP combined with various counterstaining techniques. The regions were: (i) the constitutive centromeric heterochromatin, (ii) an interstitial C-band positive insertion on chromosome 1, Is(HSR1;C5)1Lub, and (iii) the chromatin containing rDNA (designated nucleolar chromatin herein). Incorporated biotin was detected either by the horseradish peroxidase reaction with diaminobenzidine (DAB) or the alkaline phosphatase reaction with fast red. The latter resulted in a water insoluble red precipitate, which was easily removable by any organic solution thus allowing the application of various counterstaining protocols. DNase I sensitivity of the three chromatin regions was screened in different cell types of the mouse testis. The interstitial Is(HSR) region was highly DNase I sensitive when it was recognizable by strong mithramycin fluorescence. The centromeric heterochromatin was DNase I resistant when it was compacted into microscopically visible chromosomal structures (mitosis, pachytene, metaphase I and II). In interphase nuclei from Sertoli cells and spermatogonia it became highly DNase I sensitive. In round spermatids it displayed medium DNase I sensitivity. Nucleolar chromatin was not labelled by in situ nick translation when silver staining demonstrated strong protein production. Sperm cells were highly DNase I sensitive from stages 11 to 15, but resistant as mature spermatozoa.
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PMID:Nonradioactive in situ nick translation combined with counterstaining: characterization of C-band and silver positive regions in mouse testicular cells. 169 89

Secretory vesicles that accumulate in the temperature-sensitive sec6-4 strain of yeast have been shown to contain a vanadate-sensitive ATPase, presumably en route to the plasma membrane (Walworth, N. C., and Novick, P. J. (1987) J. Cell Biol. 105, 163-174). We have now established this enzyme to be a fully functional form of the PMA1 [H+]ATPase, identical in its catalytic properties to that found in the plasma membrane. In addition, the secretory vesicles are sealed tightly enough to permit the measurement of ATP-dependent proton pumping with fluorescent probes. We have gone on to exploit the vesicles as an expression system for site-directed mutants of the ATPase. For this purpose, a sec6-4 strain has been constructed in which the chromosomal PMA1 gene is under control of the GAL1 promoter; the mutant pma1 allele to be studied is introduced on a centromeric plasmid under the control of a novel heat shock promoter. In galactose medium at 23 degrees C, the wild-type ATPase is produced and supports normal vegetative growth. When the cells are switched to glucose medium at 37 degrees C, however, the wild-type gene turns off, the mutant gene turns on, and secretory vesicles accumulate. The vesicles contain a substantial amount of newly synthesized, plasmid-encoded ATPase (5-10% of total vesicle protein), but only traces of residual wild-type PMA1 ATPase and no detectable mitochondrial ATPase, vacuolar ATPase, or acid or alkaline phosphatase. To test the expression strategy, we have made use of pma1-105 (Ser368----Phe), a vanadate-resistant mutant previously characterized by standard methods (Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem. 264, 21857-21864). In secretory vesicles, as expected, the plasmid-borne pma1-105 allele gives rise to a mutant enzyme with a reduced rate of ATP hydrolysis and a 100-fold increase in Ki for vanadate. Proton pumping is similarly resistant to vanadate. Thus, the vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase. They should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.
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PMID:Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis. 182 8

Degenerate probe DNA, homologous to part of the 234-bp repeated mouse gamma (major) satellite DNA, was generated by primer-directed in vitro DNA amplification using the polymerase chain reaction with oligonucleotide primers that anneal in the most conserved parts of the repeat. Probe labeling with biotin was performed during DNA polymerization. In situ hybridization of probe DNA with metaphase chromosome preparations showed exclusive binding of probe molecules to the centromeric region of mouse chromosomes. We applied the probe DNA for labeling of mouse heterochromatin in metaphase chromosomes, as well as interphase cell nuclei, and compared results of probe visualization using avidin tagged with either fluorescein or alkaline phosphatase in combination with a chromogenic substrate.
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PMID:Non-isotopical labeling of murine heterochromatin in situ by hybridization with in vitro-synthesized biotinylated gamma (major) satellite DNA. 186 59

The Na+/H+ antiporter is a ubiquitous membrane-associated protein that plays an important role in the regulation of intracellular pH. APNH, a gene encoding the antiporter, has been cloned and mapped to the short arm of chromosome 1 by in situ hybridization. Using the polymerase chain reaction, we have amplified a 376 base pair fragment corresponding to the 5' end of APNH. We have detected a polymorphism within this fragment by denaturing gradient gel electrophoresis. Using polymorphisms at other 1p loci (ALPL, the gene for alkaline phosphatase, RH and D1S57), we have been able to map APNH telomeric to D1S57 and close to RH and ALPL by genetic linkage. APNH is a plausible candidate gene for human essential hypertension; the APNH polymorphism combined with a knowledge of its genetic map location allow this candidate to be tested in hypertensive kindreds and sib-pairs.
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PMID:The Na+/H+ antiporter: a "melt" polymorphism allows regional mapping to the short arm of chromosome 1. 197 10

Use of prometaphase chromosome preparations has led to significant improvements in the localization of both NORs and ribosomal gene clusters in the short arms of human acrocentric chromosomes. An improvement of the NOR silver-staining method, followed by trypsin-Giemsa banding, was used to identify the precise location of the NOR on each human acrocentric chromosome. For comparison, the satellite, stalk, and centromeric region were also identified with the aid of both Q- and G-banding techniques. The amount of silver impregnation present in the stalk region of the D- and G-group chromosomes was unique for each of these acrocentric chromosomes and depended on the length of the stalk. In situ hybridization was used to locate the ribosomal gene clusters. A plasmid containing 5.6 kb of the 18S rDNA gene was first oligolabeled with bio-16-dUTP, then hybridized in situ to metaphase chromosomes and visualized by an alkaline phosphatase color-detection system. Our results indicated that, in most cases, the location of the 18S rDNA gene cluster in the stalk region was indistinguishable from the site of silver impregnation. However, exceptions were noted, suggesting a multiplicity of arrangements of the ribosomal gene clusters. A model is proposed to describe the spatial relationship of NORs (transcriptional activity) and the ribosomal gene clusters.
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PMID:Visualization of NORs in relation to the precise chromosomal localization of ribosomal RNA genes. 247 78

We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.
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PMID:Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum. 318 18

A five-point linkage map has been established between the loci encoding liver/bone/kidney alkaline phosphatase (ALPL), enolase 1-alpha (ENO1), glucose-phosphate isomerase (GPI), phosphogluconate dehydrogenase (PGD), and transforming growth factor beta 1 (TGFB1) in swine. Linkage analysis was performed using the Meishan x Yorkshire three-generation reference pedigree at the University of Illinois (n = 91). Previously ENO1, GPI, PGD, and TGFB1 were mapped to porcine chromosome 6q by in situ hybridization but the linkage relations of TGFB1 and ENO1 with other loci in this group were not investigated. Based on mapping data from human chromosomes 1 and 19 and mouse chromosomes 4 and 7, it was postulated that ALPL should reside among or near these loci. Restriction fragment length polymorphisms were identified for ALPL, ENO1, and TGFB1. GPI (EC 5.3.1.9) and PGD (EC 1.1.1.44) phenotypes were determined by agarose gel electrophoresis of isozymes. Marker data were analyzed using the MLINK (two locus) and ILINK (multilocus) programs from LINKAGE (version 5.10). The most likely locus order between GPI-TGFB1-(PGD-ENO1)-ALPL with recombination rates of 0.049, 0.044, 0.000, and 0.156, respectively, could not be significantly determined. The maximum five-point lod score was the same to four decimal places irrespective of the order of ENO1 and PGD. This indicates that ENO1 and PGD are very closely linked and that ALPL is located telomeric to the established linkage group on pig chromosome 6.
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PMID:Linkage relationships between ALPL, ENO1, GPI, PGD, and TGFB1 on porcine chromosome 6. 810 72

Procedures that combine immunocytochemistry (ICC) and in situ hybridization (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapid procedures for simultaneous detection of a nuclear antigen, progesterone receptors (PR), and the centromeric region of chromosome 11 (to which the human PR gene has been assigned) in T47-D cells. Proteins were stained by precipitates of horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (APase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable contrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X-phosphate-immunostained cells with interphasic fluorescent in situ hybridization (FISH). By contrast, we combined the APase-Fast Red ICC with an immunocytochemical ISH using alkaline phosphatase-NBT-X-phosphate detection. Only the procedure combining APase-NBT-X-phosphate ICC and FISH ensures optimal visualization of both the PR content and the number of chromosome 11. This method easily provides simultaneous localization of DNA and protein targets in the same cells and should be applicable to many other situations.
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PMID:Methods for simultaneous interphase in situ hybridization and nuclear antigen immunocytochemistry in T47-D cells. 860 77

The occurrence and distribution pattern of spontaneous single-strand breaks (nicks) and/or gaps of mouse chromosomal DNA were studied with the help of nick-translation procedure omitting exogenous nucleases. The holoenzyme and a Klenow's fragment were used at a concentration of 0.I. U/20 microl of reaction mixture, resp. Bio-dUTP and streptavidin-alkaline phosphatase were used for labeling and detection. Chromosomes of postimplantation embryos and bone marrow were not stained. Chromosomes of all preimplantation stages of development were homogeneously stained with prominent dots of various size and intensities of grayish. DNA Pol I and the Klenow enzyme demonstrated a similar pattern of labelling. The centromeric heterochromatin was not labeled. The label was localized asymmetrically exclusive of NOR and telomeric regions.
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PMID:[Single-stranded DNA breaks in the chromosomes of early mouse embryos]. 865 75

Terminal segments (telomeres) of linear mitochondrial DNA (mtDNA) molecules of the yeast Candida parapsilosis consist of large sequence units repeated in tandem. The extreme ends of mtDNA terminate with a 5' single-stranded overhang of about 110 nucleotides. We identified and purified a mitochondrial telomere-binding protein (mtTBP) that specifically recognizes a synthetic oligonucleotide derived from the extreme end of this linear mtDNA. MtTBP is highly resistant to protease and heat treatments, and it protects the telomeric probe from degradation by various DNA-modifying enzymes. Resistance of the complex to bacterial alkaline phosphatase suggests that mtTBP binds the very end of the molecule. We purified mtTBP to near homogeneity using DNA affinity chromatography based on the telomeric oligonucleotide covalently bound to Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fractions revealed the presence of a protein with an apparent molecular mass of approximately 15 kDa. UV cross-linking and gel filtration chromatography experiments suggested that native mtTBP is probably a homo-oligomer. MtTBP of C. parapsilosis is the first identified protein that specifically binds to telomeres of linear mitochondrial DNA.
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PMID:Identification of a putative mitochondrial telomere-binding protein of the yeast Candida parapsilosis. 900 55


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