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The entire cDNA encoding the large subunit of mouse DNA polymerase δ (mPolδ; EC 2.7.7.7) has been cloned and expressed in various bacterial expression systems. A soluble protein could only be obtained when mPolδ was produced as a glutathione S-transferase (GST) fusion protein and the incubation temperature of the expression strain was reduced to 30°C. After purification over a glutathione-Sepharose...
Ten mutator alleles of MIP1, the gene encoding mitochondrial (mt) DNA polymerase, have been isolated after in vitro random mutagenesis. Five mutations causing a 100-400-fold increase in the frequency of erythromycin-resistant (Er R ) mt mutants in yeast mapped to the 3 -5 exonuclease (Exo) domain, and mainly to the three conserved motifs Exo1, Exo2 and Exo3 of this domain, highlighting...
We have purified, by sequence-specific affinity chromatography, a mitochondrial (mt) matrix protein which binds to the curved DNA located between the replication origin (ori) of the leading strand (ori-H) and the two transcription promoters in the rat mt genome. The protein was characterized by gel electrophoresis as a 67-kDa polypeptide and seems to be involved in the DNA contact on the mt light...
In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm). We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig. The Rm lig gene encodes a protein...
A putative full-length 1.7-kb cDNA, encoding a murine protein that specifically binds to the nonamer portion of the V(D)J recombinational signal sequence (RSS) element, has been cloned. By its sequence analysis, this cDNA is identical to a portion of the 4.5-kb murine replication factor C large-subunit-encoding cDNA. By Northern blot analysis, the 1.7-kb mRNA species is observed in murine immature...
Part of the topoisomerase I (TopoI)-encoding gene from Plasmodium falciparum (Pf) was isolated by PCR from cDNA using oligodeoxyribonucleotides modelled on the highly conserved regions of sequence from other species. The entireTopoI gene was obtained by screening a Pf K1 HindIII-EcoRI genomic library in λNM1149 with a random-labeled heterologous probe from the Saccharomyces cerevisiae TopoI gene...
The polA gene (encoding DNA polymerase I) from Mycobacterium tuberculosis was cloned using an internal gene segment probe generated by PCR amplification of genomic DNA [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide 904 amino acids (aa) in length that shares 89% identity with a 911-aa homologue fromMycobacterium leprae . The polypeptide has all of the primary structural...
The cDNA encoding the catalytic subunit of Drosophila melanogaster (Dm) DNA polymerase δ (Polδ) was isolated by a combination of PCR amplification and cDNA library screening. The cDNA is 3457 nucleotides in length and contains an open reading frame (ORF) that encodes a protein of 1092 amino acids (124 799 Da). The ORF contains the sequence that was determined for a peptide from the purified catalytic...
We have identified the direction of replication of a bacteriophage P1 lytic replicon. This was accomplished by constructing λP1 lysogens that contain a functional P1 lytic replicon and analysing which of two nearby bacterial DNA markers flanking the λ prophage were amplified when that replicon was activated. We demonstrate that both DNA markers are coordinately amplified, a result consistent with...
We have cloned, sequenced and characterized the gene encoding a DNA polymerase from the thermoacidophilic archaeonSulfolobus acidocaldarius (Sac). The putative transcription promoter and terminator elements, as well as a potential ribosome-binding site (rbs), have been identified in the flanking regions. One large open reading frame (ORF) found in the sequenced portion of the Sac genome encodes...
DNA topoisomerase I is an enzyme involved in several processes related to DNA metabolism. Despite the physiological importance, the regulation of top1 gene expression has not yet been investigated in plants. In order to monitor the possible correlation between levels of top1 transcripts and the proliferative state of the cell, two partially overlapping cDNAs encoding DNA topoisomerase I from Daucus...
In Saccharomyces cerevisiae, the CDC2 gene encodes the large subunit of DNA polymerase III, the analogue of mammalian DNA polymerase δ. We have isolated DNA fragments from a library of Candida albicans genomic DNA in the vector pRS316 that rescue temperature sensitive cdc2 mutations in S. cerevisiae. These fragments contain an ORF coding for a protein of 1038 aa with a predicted molecular mass of...
We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide...
MCM (minichromosome maintenance) gene family of Saccharomyces cerevisiae encodes essential DNA replication factors that participate in the initiation of DNA replication. In addition, their localization to the nucleus in a mitosis-dependent manner fueled the hypothesis that MCMs also act to couple DNA replication to mitosis. We report the identification of a Drosophila gene family with extensive...
A large portion of the consensus sequence of four mammalian involucrin genes comprises four pairs of complementary oligonucleotides. One of the oligonucleotides in each pair is rich in purines and the other in pyrimidines. The perfect complementarity of a number of pairs apparently resulted from a concerted single nucleotide substitution in each pairing oligonucleotide from their respective consensus...
Genomes comprising a pair of separated inverted repeats and called `amphimers' are reviewed. Amphimeric genomes are observed in a large variety of different organisms, ranging from archaebacteria to mammals. The widespread existence of amphimeric genomes in nature could be due to their particular dynamic structure. Amphimeric genomes containing long inverted segments may provide the only form in which...
The complete nucleotide sequence of the B. subtilis bacteriophage SPP1 is described. The genome is 44 007 bp in size and has a base composition of 43.7% dG + dC. Only 32.2 kb are essential for phage amplification under laboratory conditions. Transcription using only the heavy strand is asymmetric. Eighty-one orfs organized in five early and four late operons were identified. Experiments have...
The Cdc7 protein kinase of Saccharomyces cerevisiae is a critical regulator of several aspects of DNA metabolism and cell cycle progression. We describe the isolation of a human gene encoding a Cdc7 homolog. The Cdc7 Hs protein sequence is 27% identical to that of the yeast protein, includes features unique to yeast Cdc7, and contains all conserved catalytic residues of protein kinases...
Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant, p53 has been shown to be involved in DNA replication and DNA repair...
We present a computer model to predict the patterns expected for the replication intermediates (RIs) of DNA fragments analyzed by neutral/neutral two-dimensional (2D) agarose gel electrophoresis. The model relies on the mode of replication (uni- or bi-directional), the electrophoretic mobility of linear DNA fragments and the retardation caused by the three-dimensional shape of non-linear molecules...
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