A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii. (1/1105)

In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity.  (+info)

Structural organization and expression of the mouse gene for Pur-1, a highly conserved homolog of the human MAZ gene. (2/1105)

We have characterized the genomic structure and expression of the mouse gene for Pur-1. The cloned Pur-1 gene spans a 5-kb region encompassing the promoter, five exons, four introns and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. The promoter region has typical features of a housekeeping gene: a high G + C content (77.5%); a high frequency of CpG dinucleotides, in particular within the region 0.5 kb upstream of the site of initiation of translation; and the absence of canonical TATA and CAAT boxes. S1 nuclease protection assay demonstrated the presence of multiple sites for initiation of transcription around a site 108 nucleotides upstream of the ATG codon. Comparison of Pur-1 with the human gene for MAZ (Myc-associated zinc finger protein) revealed a striking homology of both their nucleotide and deduced protein sequences, an identical genomic organization and high similarity in promoter architecture and mRNA expression pattern. Sequence analysis of the 5'-flanking region of Pur-1 revealed numerous potential binding sites for transcription factors Sp1, AP-2 and Pur-1/MAZ itself. An element required for basal Pur-1 expression was mapped from nucleotide -258 to +43. This region also mediated stimulation of basal transcription by ectopically expressed MAZ protein. We conclude that the Pur-1 gene is the murine homolog of human MAZ and, like it, belongs to the family of housekeeping genes.  (+info)

Cloning and characterization of a mammalian pseudouridine synthase. (3/1105)

This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame.  (+info)

Glycogen synthase phosphatase interacts with heat shock factor to activate CUP1 gene transcription in Saccharomyces cerevisiae. (4/1105)

Upon heat shock, transcription of many stress-inducible genes is rapidly and dramatically stimulated by heat shock factor (HSF). A central region of the yeast HSF (designated HSFrr for "repression region") was previously identified and proposed to be involved in repressing the activation domain under non-heat-shock conditions. Here, we used the phage display system to isolate proteins that interact with HSFrr. This should identify factors that modulate HSF activity or directly participate in HSF-mediated transcriptional activation. We constructed a randomly sheared yeast genomic library to express yeast proteins on the surface of lambda phage. HSFrr binding phages were selected by cycles of affinity chromatography. DNA sequencing identified an HSFrr-interacting phage that contains the GAC1 gene. The GAC1 gene encodes the regulatory subunit for a type 1 serine/threonine phosphoprotein phosphatase, Glc7. Both gac1 and glc7 mutations had little effect on HSF activation of gene transcription of two heat shock genes, SSA4 and HSP82. In contrast, heat shock induction of CUP1 gene expression was completely abolished in a glc7 mutant and reduced in a gac1 mutant. The results demonstrate that the Glc7 phosphatase and its Gac1 regulatory subunit play positive roles in HSF activation of CUP1 transcription.  (+info)

DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959). (5/1105)

A total of 48 pathovars of Pseudomonas syringae and eight related species were studied by DNA-DNA hybridization (S1 nuclease method) and ribotyping. The existence of nine discrete genomospecies was indicated. Genomospecies 1 corresponded to P. syringae sensu stricto and included P. syringae pathovars syringae, aptata, lapsa, papulans, pisi, atrofaciens, aceris, panici, dysoxyli and japonica. Genomospecies 2 included P. syringae pathovars phaseolicola, ulmi, mori, lachrymans, sesami, tabaci, morsprunorum, glycinea, ciccaronei, eriobotryae, mellea, aesculi, hibisci, myricae, photiniae and dendropanacis and nomenspecies Pseudomonas savastanoi, Pseudomonas ficuserectae, Pseudomonas meliae and Pseudomonas amygdali, which are thus synonymous. P. amygdali is the earliest valid name for this genomospecies. Genomospecies 3 included P. syringae pathovars tomato, persicae, antirrhini, maculicola, viburni, berberidis, apii, delphinii, passiflorae, philadelphi, ribicola and primulae. We recommend strain CFBP 2212 of P. syringae pv. tomato to serve as the type strain. Genomospecies 4 included 'Pseudomonas coronafaciens' and P. syringae pathovars porri, garcae, striafaciens, atropurpurea, oryzae and zizaniae and corresponds to 'P. coronafaciens'. Genomospecies 5 included P. syringae pv. tremae and corresponds to Pseudomonas tremae sp. nov. Genomospecies 6 included Pseudomonas viridiflava and the presently misidentified pathotype strains of P. syringae pv. ribicola and P. syringae pv. primulae and thus corresponds to P. viridiflava. Genomospecies 7 included P. syringae pv. tagetis and P. syringae pv. helianthi. We recommend strain CFBP 1694 of P. syringae pv. tagetis to serve as a reference strain. Genomospecies 8 included P. syringae pv. these and Pseudomonas avellanae and thus corresponds to P. avellanae. Genomospecies 9 included P. syringae pv. cannabina and corresponds to Pseudomonas cannabina sp. nov. Ribotyping (SmaI and HincII endonucleases) could separate seven of the nine genomospecies. The unnamed genomospecies 3 and 7 will be named when phenotypic data are available for identification. Two species are described, P. tremae sp. nov. and P. cannabina sp. nov. Other species will be named when phenotypic data are available for identification.  (+info)

Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader Streptomyces reticuli. (6/1105)

Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular beta-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the ceb operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in S. reticuli and Streptomyces lividans. Transcription of polycistronic cebEFG and bglC mRNAs is induced by cellobiose, whereas the cebR gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of S. reticuli, since insertional inactivation of the cebE gene prevents high-affinity uptake of cellobiose.  (+info)

Characterisation of holoenzyme lacking sigmaN regions I and II. (7/1105)

The sigma-N (sigmaN) protein associates with bacterial core RNA polymerase to form a holoenzyme that is silent for transcription in the absence of enhancer-binding activator proteins. Here we show that the acidic Region II of sigmaN from Klebsiella pneumoniae is dispensable for polymerase isomerisation and trans-cription under conditions where the inhibited state of the holoenzyme is relieved by removal of sigmaN Region I sequences. Holoenzymes lacking Region I or Regions I+II were equally susceptible to the order of addition-dependent inhibition or stabilisation of DNA binding afforded by in trans Region I sequences. Region I+II-deleted [sigma] formed a holoenzyme with a DNA-binding activity more susceptible to inhibition by non-specific DNA than that lacking Region I. Region II sequences appear more closely associated with formation of a holoenzyme and [sigma] proficient in DNA binding than with changes in holoenzyme conformation needed for unmasking a single-strand DNA-binding activity used for open complex for-mation. Region II may therefore function to optimise DNA interactions for an efficient sigma cycle.  (+info)

Assay for reactive oxygen species-induced DNA damage: measurement of the formamido and thymine glycol lesions. (8/1105)

A 32P-postlabeling assay has been developed for the simultaneous detection of the thymine glycol lesion and the formamido remnant of pyrimidine bases in DNA exposed to reactive oxygen species (ROS). The formamido lesion is a principal lesion produced in X-irradiated DNA oligomers when oxygen is available to mediate the damage process. Production of the well-known thymine glycol lesion is less dependent on the concentration of oxygen. These two lesions have the common property that they make the phosphoester bond 3' to the modified nucleoside resistant to hydrolysis by nuclease P1. Our assay uses 32P-postlabeling to measure these lesions in the form of modified dimers obtained from DNA by nuclease P1 digestion. Appropriate carriers and internal standards have been chemically synthesized to improve the reliability and accuracy of the assay. The measurements were accomplished on 1-microgram samples of DNA.  (+info)