Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5ƀ upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

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Structure of active chromatin: higher-order folding of transcriptionally active chromatin in control and hypothyroid rat liver

Biochemical Journal ◽

10.1042/bj3220289 ◽

1997 ◽

Vol 322 (1) ◽

pp. 289-296 ◽

Cited By ~ 1

Author(s):

Kulbhushan TIKOO ◽

Q. Anwar HAMID ◽

Ziledar ALI

Keyword(s):

Ionic Strength ◽

Rat Liver ◽

Sedimentation Rate ◽

Higher Order ◽

Micrococcal Nuclease ◽

Percentage Increase ◽

Active Chromatin ◽

Nuclease Digestion ◽

Rate Of Sedimentation ◽

Cd Studies

Investigations have been carried out into the salt-induced higher-order folding in the transcriptionally active chromatin region of rat liver nuclei by nuclease digestion, sedimentation and CD. The sensitivity of active chromatin in nuclei to micrococcal nuclease was suppressed by raising the ionic strength from 25 to 90 mM, indicating the occurrence of salt-induced condensation. The rate of sedimentation of fractionated inactive chromatin fragments of both moderate (∼3.5 kbp) and large (∼8.8 kbp) size increased maximally to the same extent, while that of active chromatin fragments was dependent on their size. The rate of sedimentation of moderately sized active chromatin fragments (∼5.5 kbp) showed a maximal 15% increase at 90 mM ionic strength. In contrast, a large increase (at least 60%) in the sedimentation rate of large active chromatin fragments (∼21 kbp) was observed at 65 mM ionic strength. A reasonable degree of higher-order folding was observed in large active chromatin fragments even at 25 mM ionic strength. On considering the percentage increase in sedimentation rate as a measure of the higher-order folding of chromatin, a different type of higher-order folding was observed in active chromatin fragments. Although the percentage increase in sedimentation decreased from 40 to 24% with an increase in the size of active chromatin from ∼3 to ∼9 kbp, a further increase in size up to 16 kbp brought the percentage increase back to 40%. CD studies agreed with the conclusions drawn from sedimentation studies. Active chromatin from hypothyroid rats showed similar folding behaviour, but the order of folding was slightly lower than for control active chromatin, at all sizes.

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Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family

Biochemical Journal ◽

10.1042/bj2870261 ◽

1992 ◽

Vol 287 (1) ◽

pp. 261-267 ◽

Cited By ~ 39

Author(s):

C T Dolphin ◽

E A Shephard ◽

S Povey ◽

R L Smith ◽

I R Phillips

Keyword(s):

Blot Hybridization ◽

Southern Blot Hybridization ◽

Chromosome 1 ◽

Cdna Clones ◽

Amino Acid Residues ◽

Primary Sequence ◽

Gene Encoding ◽

Isolation And Characterization ◽

Distinct Member ◽

Human Chromosome 1

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

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Isolation and characterization of mutants of the citrus canker pathogen Xanthomonas axonopodis pv. citri that induce a distinct pattern of disease

Canadian Journal of Botany ◽

10.1139/b00-078 ◽

2000 ◽

Vol 78 (8) ◽

pp. 1002-1009

Author(s):

Shu Yun Tung ◽

Tsong Teh Kuo

Keyword(s):

Blot Hybridization ◽

Random Mutagenesis ◽

Southern Blot Hybridization ◽

Citrus Canker ◽

Distinct Pattern ◽

Wild Type ◽

In Planta ◽

Xanthomonas Axonopodis ◽

Isolation And Characterization ◽

Indigenous Plasmids

Random mutagenesis with the transposon Tn5tac1 in Xanthomonas axonopodis pathovar citri, the causal agent of citrus canker, generated four mutants with altered pathogenicity. These mutants were classified into three groups: (i) the nonpathogenic (NP) mutants XT10 and XT122, which did not induce any visible symptoms in the host; (ii) the WS- mutant XT27, which induced a callus-like lesion but not a watersoaked lesion; and (iii) the CL- mutant XT37, which was unable to induce a callus-like eruption but did induce the formation of a watersoaked lesion around the infection site. The NP mutants failed to grow in planta, whereas the WS- and CL- mutants showed a reduced growth rate relative to that of the wild type. Co-inoculation of leaves with the WS- and CL- mutants did not result in complementation of their respective defects. The extent of extracellular accumulation of polysaccharide, protease, and amylase activities by each of the mutants was similar to that of the wild type. The extracellular activity of polygalacturonate lyase of XT27 was reduced relative to that of the wild type and other mutants. Unlike the wild type and other mutants, XT27 also required glutamic acid for growth in culture. Southern blot hybridization revealed that each of the mutants resulted from transposon insertion at a single site; the insertion sites for XT10 and XT27 were located in the chromosome, whereas those for XT37 and XT122 were located in the indigenous plasmids. These results provide evidence that bacterial genes contribute independently to the pathogenesis of citrus canker.Key words: citrus canker, pathogenicity genes, transposon mutagenesis.

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Glucose-6-phosphate dehydrogenase of malaria parasite Plasmodium falciparum

Blood ◽

10.1182/blood.v69.5.1528.bloodjournal6951528 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1528-1530

Author(s):

A Yoshida ◽

EF Jr Roth

Keyword(s):

Plasmodium Falciparum ◽

Malaria Parasite ◽

Blot Hybridization ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Minor Component ◽

Southern Blot Hybridization ◽

Starch Gel Electrophoresis ◽

Parasite Plasmodium Falciparum ◽

A Minor ◽

Glucose 6 Phosphate Dehydrogenase

Plasmodium falciparum growth is impaired in glucose-6-phosphate dehydrogenase (G6PD)-deficient red blood cells (RBCs), and malaria has been implicated in the spreading of deficient variants in malaria- endemic areas. Recent reports suggest that the malaria parasite can adapt itself to grow in these variant RBCs by producing its own G6PD, but studies on parasite G6PD are very limited. In this report, we define the properties of the parasite G6PD. G6PD was partially purified from infected and uninfected variant RBCs associated with severe G6PD deficiency. G6PD from infected RBCs contained two components separable by starch gel electrophoresis: a major component (approximately 90% activity) with a very slow anodal electrophoretic mobility and a minor component (approximately 10% activity) with the same mobility as the host G6PD. Parasite G6PD exhibited much higher affinity (low Km) to G6P and nicotinamide-adenine dinucleotide phosphate (NADP) than did human G6PD. Southern blot hybridization indicated that the parasite genome contained nucleotide sequences that were hybridizable with the human G6PD cDNA. These data indicate that the parasite is capable of adapting to G6PD-deficient RBCs by producing its own G6PD.

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Glucose-6-phosphate dehydrogenase of malaria parasite Plasmodium falciparum

Blood ◽

10.1182/blood.v69.5.1528.1528 ◽

1987 ◽

Vol 69 (5) ◽

pp. 1528-1530 ◽

Cited By ~ 17

Author(s):

A Yoshida ◽

EF Jr Roth

Keyword(s):

Plasmodium Falciparum ◽

Malaria Parasite ◽

Blot Hybridization ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Minor Component ◽

Southern Blot Hybridization ◽

Starch Gel Electrophoresis ◽

Parasite Plasmodium Falciparum ◽

A Minor ◽

Glucose 6 Phosphate Dehydrogenase

Abstract Plasmodium falciparum growth is impaired in glucose-6-phosphate dehydrogenase (G6PD)-deficient red blood cells (RBCs), and malaria has been implicated in the spreading of deficient variants in malaria- endemic areas. Recent reports suggest that the malaria parasite can adapt itself to grow in these variant RBCs by producing its own G6PD, but studies on parasite G6PD are very limited. In this report, we define the properties of the parasite G6PD. G6PD was partially purified from infected and uninfected variant RBCs associated with severe G6PD deficiency. G6PD from infected RBCs contained two components separable by starch gel electrophoresis: a major component (approximately 90% activity) with a very slow anodal electrophoretic mobility and a minor component (approximately 10% activity) with the same mobility as the host G6PD. Parasite G6PD exhibited much higher affinity (low Km) to G6P and nicotinamide-adenine dinucleotide phosphate (NADP) than did human G6PD. Southern blot hybridization indicated that the parasite genome contained nucleotide sequences that were hybridizable with the human G6PD cDNA. These data indicate that the parasite is capable of adapting to G6PD-deficient RBCs by producing its own G6PD.

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Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽

10.1099/mic.0.26778-0 ◽

2004 ◽

Vol 150 (4) ◽

pp. 967-978 ◽

Cited By ~ 25

Author(s):

C. Viana-Niero ◽

P. E. de Haas ◽

D. van Soolingen ◽

S. C. Leão

Keyword(s):

Mycobacterium Tuberculosis ◽

Phospholipase C ◽

Genetic Polymorphisms ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Clinical Isolates ◽

Significant Similarity ◽

Genomic Deletions ◽

Genes Encoding ◽

Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

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