TATA-binding protein and nuclear differentiation in Tetrahymena thermophila.

Unambiguous TATA boxes have not been identified in upstream sequences of Tetrahymena thermophila genes analyzed to date. To begin a characterization of the promoter requirements for RNA polymerase II, the gene encoding TATA-binding protein (TBP) was cloned from this species. The derived amino acid sequence for the conserved C-terminal domain of Tetrahymena TBP is one of the most divergent described and includes a unique 20-amino-acid C-terminal extension. Polyclonal antibodies generated against a fragment of Tetrahymena TBP recognize a 36-kDa protein in macronuclear preparations and also cross-react with yeast and human TBPs. Immunocytochemistry was used to examine the nuclear localization of TBP during growth, starvation, and conjugation (the sexual phase of the life cycle). The transcriptionally active macronuclei stained at all stages of the life cycle. The transcriptionally inert micronuclei did not stain during growth or starvation but surprisingly stained with anti-TBP throughout early stages of conjugation. Anti-TBP staining disappeared from developing micronuclei late in conjugation, corresponding to the onset of transcription in developing macronuclei. Since micronuclei do not enlarge or divide at this time, loss of TBP appears to be an active process. Thus, the transcriptional differences between macro- and micronuclei that arise during conjugation are associated with the loss of a major component of the basal transcription apparatus from developing micronuclei rather than its appearance in developing macronuclei.

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Cloning and Characterization of an EssentialSaccharomyces cerevisiaeGene,TAF40, Which Encodes yTAFII40, an RNA Polymerase II-specific TATA-binding Protein-associated Factor

Journal of Biological Chemistry ◽

10.1074/jbc.272.14.9436 ◽

1997 ◽

Vol 272 (14) ◽

pp. 9436-9442 ◽

Cited By ~ 20

Author(s):

Edward R. Klebanow ◽

David Poon ◽

Sharleen Zhou ◽

P. Anthony Weil

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Binding Protein ◽

Tata Binding Protein ◽

Associated Factor

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RNA Polymerase II Localizes in Tetrahymena thermophila Meiotic Micronuclei When Micronuclear Transcription Associated with Genome Rearrangement Occurs

Eukaryotic Cell ◽

10.1128/ec.3.5.1233-1240.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1233-1240 ◽

Cited By ~ 34

Author(s):

Kazufumi Mochizuki ◽

Martin A. Gorovsky

Keyword(s):

Life Cycle ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genome Rearrangement ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Essential Gene ◽

Gene Encoding ◽

Sexual Process ◽

Rnap Ii

ABSTRACT The germ line micronucleus in Tetrahymena thermophila is transcriptionally silent in vegetatively growing cells. However, micronuclear transcription has been observed in the early (“crescent”) stages of the sexual process, conjugation. This transcription is proposed to play a central role in identifying sites for subsequent genome rearrangements that accompany development of the somatic macronucleus from the micronucleus. RPB3 (cnjC), a gene encoding a protein homologous to the third largest subunit of RNA polymerase II (RNAP II), was previously reported to be expressed specifically during conjugation, suggesting a role in micronucleus-specific transcription. Rpb3p localized in the micronucleus only during the meiotic prophase, when micronuclear transcription occurs, and its intranuclear distribution is strikingly similar to that for previously described sites of micronuclear RNA synthesis. By contrast, Rpc5p, the homologous subunit shared by RNAPs I and III, was not detectable in the micronucleus at any stage of the life cycle. However, Rpb3p is not specific to the transcribing micronucleus. Like Rpc5p, it also localizes to macronuclei in all stages of the life cycle. Rpb3p is encoded by a unique, essential gene in Tetrahymena. Thus, RNAP II is associated with both somatic transcription and crescent transcription and probably has an important role in genome rearrangement.

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Biochemical Characterization of the TATA-binding Protein-Gal4 Activation Domain Complex

Journal of Biological Chemistry ◽

10.1074/jbc.m003760200 ◽

2000 ◽

Vol 275 (41) ◽

pp. 31914-31920 ◽

Cited By ~ 12

Author(s):

Yueqing Xie ◽

Carilee Denison ◽

Sang-Hwa Yang ◽

David A. Fancy ◽

Thomas Kodadek

Keyword(s):

Biochemical Characterization ◽

Binding Protein ◽

Tata Binding Protein ◽

Activation Domain ◽

Gal4 Activation Domain

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Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

Journal of Bacteriology ◽

10.1128/jb.186.15.4885-4893.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4885-4893 ◽

Cited By ~ 154

Author(s):

Takane Katayama ◽

Akiko Sakuma ◽

Takatoshi Kimura ◽

Yutaka Makimura ◽

Jun Hiratake ◽

...

Keyword(s):

Amino Acid ◽

Genomic Library ◽

Bifidobacterium Bifidum ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Amino Acid Residues ◽

Gene Encoding ◽

Sugar Chain ◽

Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

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Characterization of a Sinorhizobium melilotiATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake

Journal of Bacteriology ◽

10.1128/jb.182.13.3717-3725.2000 ◽

2000 ◽

Vol 182 (13) ◽

pp. 3717-3725 ◽

Cited By ~ 31

Author(s):

Eric Boncompagni ◽

Laurence Dupont ◽

Tam Mignot ◽

Magne Østeräs ◽

Annie Lambert ◽

...

Keyword(s):

Glycine Betaine ◽

Sinorhizobium Meliloti ◽

Binding Protein ◽

Compatible Solutes ◽

Site Directed Mutagenesis ◽

Uptake System ◽

Periplasmic Binding Protein ◽

Gene Encoding ◽

Inhibition Of Growth

ABSTRACT The symbiotic soil bacterium Sinorhizobium melilotiuses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli(ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed inhut mutants (hutX and hutH2). Expression analysis of the hut operon determined using ahutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

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Molecular characterization of SerH3, a Tetrahymena thermophila gene encoding a temperature-regulated surface antigen

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.6091-6096.1990 ◽

1990 ◽

Vol 10 (11) ◽

pp. 6091-6096

Author(s):

M M Tondravi ◽

R L Willis ◽

H D Love ◽

G A Bannon

Keyword(s):

Surface Antigen ◽

Dna Sequences ◽

Tetrahymena Thermophila ◽

Transcription Unit ◽

Genomic Fragment ◽

Gene Encoding ◽

Tryptophan Residues ◽

Functional Copy ◽

Regular Patterns

The DNA sequences of a cDNA clone and the macronuclear genomic fragment corresponding to the functional copy of the SerH3 surface antigen gene of Tetrahymena thermophila were determined. Primer extension and nuclease protection assays show that the SerH3 transcription unit is 1,425 nucleotides long and contains no introns. The predicted polypeptide encoded by the SerH3 gene has a molecular mass of 44,415 daltons; one-third of its 439 residues are either cysteine, serine, or threonine. The central half of the polypeptide consists of three homologous domains in tandem array; within these domains, the cysteine, proline, and tryptophan residues occur in highly regular patterns.

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The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of the nirAGene and Characterization of the Recombinant Enzyme

Journal of Bacteriology ◽

10.1128/jb.181.8.2323-2329.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2323-2329 ◽

Cited By ~ 25

Author(s):

Miguel Prudêncio ◽

Robert R. Eady ◽

Gary Sawers

Keyword(s):

Amino Acid ◽

Nitrite Reductase ◽

Recombinant Enzyme ◽

Leader Peptide ◽

Resonance Spectroscopy ◽

Gene Encoding ◽

Blue Copper

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

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