Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.
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PMID:Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast. 166 Jul 26

Two new linear plasmids, pK192L (4.9 kb) and pK192S (2.4 kb), were isolated from a Kluyveromyces lactis killer strain carrying pGKL1 and pGKL2. pK192L was a deletion plasmid of pGKL1, derived from a part of the ORF1, and had a palindrome structure of a 215 bp unique sequence flanked by 2.35 bp inverted repeats. pK192S was a hairpin plasmid produced by self-annealing of a single-stranded pK192L DNA. In genetic analysis, pK192L and pK192S always coexisted and replicated in cells harboring pGKL2 and pGKL1, in contrast to other pGKL1-derived deletion plasmids, such as F1, F2 and pGKLIS, which could replicate in cells carrying pGKL2 only. Based on these and other lines of evidence, it was concluded that the reason for the pGKL1 dependent replication of the pK192L/S plasmids was the absence of the intact pGKL1-ORF1 gene and that the ORF1 function was necessary for the replication of the pGKL1 genome. This finding is in good agreement with a recent view reporting that ORF1 may encode a DNA polymerase of pGKL1. In a separate experiment, four new linear plasmids were isolated from a Saccharomyces cerevisiae strain carrying pGKL1 and pGKL2. Structural analysis showed that they consisted of two pairs of hairpin-palindrome type plasmids, each derived from different parts of pGKL2, respectively. pGKL1 stability replicated in cells carrying both these pGKL2 derived deletion plasmids.
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PMID:Replication and maintenance of the Kluyveromyces linear pGKL plasmids. 306 Mar 67

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.
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PMID:Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development. 808 46

We have determined the nucleotide sequence of 2676 bp at the left part of the linear genome of Bacillus subtilis bacteriophage GA-1. Computer analysis revealed that this fragment contains two open reading frames (ORFs), ORF1 and ORF2, which contain 265 and 578 codons, respectively. Data base search revealed that ORF1 and ORF2 might encode proteins similar to the terminal protein (TP) and DNA polymerase, respectively, of bacteriophage phi29. By using extracts of B. subtilis infected with GA-1, we demonstrated that GA-1 DNA replication occurs by a protein-priming mechanism in which these two viral proteins are involved. Butylphenyl dGTP (BuPdGTP), a specific inhibitor of eukaryotic-type (family B) DNA polymerases, inhibited both the protein-primed initiation step and DNA polymerization during GA-1 DNA replication. These results suggest the involvement of a eukaryotic-type DNA polymerase, probably the product of the viral ORF2, in both stages of a replication process in which the TP primes replication at both DNA ends (replication origins). Using synthetic oligonucleotides, we carried out a mutational analysis of the GA-1 DNA right end to determine the initiation site for replication. The results indicate that initiation of replication mainly occurs opposite the second nucleotide at the 3' end of the template, although the third nucleotide can be used as an alternative initiation site. As in other TP-containing genomes, a sliding-back mechanism is proposed to account for the maintenance of the DNA length at the GA-1 DNA ends.
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PMID:Functional characterization of the genes coding for the terminal protein and DNA polymerase from bacteriophage GA-1. Evidence for a sliding-back mechanism during protein-primed GA-1 DNA replication. 896 97

Both the linear plasmids, pDHL1 (8.4 kb) and pDHL2 (9.2 kb), of Debaryomyces hansenii TK require the presence of a third linear plasmid pDHL3 (15.0 kb) in the same host cell for their replication. A 3.5 kb Bam HI-PstI fragment of pDHL1 strongly hybridized by Southern analysis to the 3.5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of this conserved region in the replication of the two smaller pDHL plasmids. The 4.2 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced. The results showed that the cloned pDHL1 fragment encodes a protein of 1000 amino acid residues, having a strong similarity to the DNA polymerase coded for by ORF1 of the killer plasmid pGKL1 from Kluyveromyces lactis. The catalytic and proof-reading exonuclease domains as well as terminal protein motif were well conserved as in DNA polymerases of pGKL1 and other yeast linear plasmids. Analysis of the cloned fragment further showed that pDHL1 encodes a protein partly similar to the alpha subunit of the K. lactis killer toxin, although killer activity was not known in the DHL system. Analysis of the 5' non-coding region of the two above pDHL1-ORFs reveal the presence of the upstream conserved sequence similar to that found upstream of pGKL1-ORFs. The possible hairpin loop structure was also found just in front of the ATG start codon of the pDHL1-ORFs like pGKL1-ORFs. Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K. lactis plasmids.
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PMID:The linear plasmid pDHL1 from Debaryomyces hansenii encodes a protein highly homologous to the pGKL1-plasmid DNA polymerase. 920 Aug 11

The nucleotide (nt) sequence of the Treponema denticola (Td) DNA gyrase beta-subunit gene (gyrB) has been determined. Southern blot analysis of Td chromosomal DNA indicated that gyrB is present as a single copy. Approximately 3.2kb of the nt sequence 5' and 0.7kb of nucleotide sequence 3' of gyrB were obtained. Analysis of the deduced amino acid (aa) sequence revealed two complete open reading frames (ORFs) (ORF1 and ORF3) and a truncated ORF (ORF4'). ORF1 has no homology to sequences in the databases, whereas ORF3 and ORF4' have significant homology to several bacterial DnaA (replication initiator) and DnaE (DNA polymerase III) proteins respectively. RT-PCR data showed that orf1-gyrB are co-transcribed, while dnaA-dnaE are co-transcribed but in the opposite direction. These data indicated that the gene organization of the Td gyrB region is unique compared with that of other bacteria. Eighteen putative DnaA boxes with several AT-rich regions were identified in the dnaA-dnaE intergenic region, and three putative DnaA boxes were identified in the gyrB-dnaA intergenic region. Spontaneous coumermycin A(1)-resistant Td mutants were isolated and characterized. The mutants have a >20-fold higher resistance to coumermycin A(1) than wild-type Td. A single point mutation in gyrB that changed GyrB Lys(136) to Glu or Thr appears to be responsible for the coumermycin A(1) resistance.
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PMID:Molecular characterization of the gyrB region of the oral spirochete, Treponema denticola. 1094 May 64

Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae. Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats. Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization. The Pichia element has four ORFs that account for 95% of the sequence. ORF1 is homologous to the putative immunity gene of the K. lactis system. A viral B-type DNA polymerase is encoded by ORF2. The predicted product of ORF3 displays similarities to the alpha- and beta-subunits of the heterotrimeric K. lactis killer toxin, also known as zymocin. A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the alpha-subunit of zymocin were identified in Orf3p. Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the alpha-subunit of K. lactis zymocin. Disruption of the major chitin synthase-gene ( CHS3) renders S. cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P. inositovora toxin. Orf4p of pPin1-3 displays only weak similarities to the gamma-subunit of zymocin, which causes a G1 cell-cycle arrest in S. cerevisiae. However, disruption of the S. cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P. inositovora toxin also. Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.
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PMID:Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora. 1368 Mar 68

Bombyx mori parvo-like virus (BmPLV) has two complementary single-stranded DNA genome (VD1 and VD2) and owns a self-encoding DNA polymerase motif, but its replication mechanism is unclear. In our previous research, a protein encoded by VD1-ORF1 was indentified in the midgut of BmPLV China Zhenjiang isolate-(BmPLV-Z) infected silkworm larvae via two-dimensional gel electrophoresis (2-DE). This protein was named as non-structural protein 2 (NS2), which showed no similarity to that of parvoviruses. To date, little is known about it. In this study, sequence alignment results showed that NS2 shared homology with some chromosomal replication initiator protein dnaA and DNA-binding response regulators. The ns2 was cloned and expressed in E. coli, and then a polyclonal antibody of the NS2 protein was prepared successfully. The data from real-time quantitative PCR displayed that the transcription of VD1-ORF1 from BmPLV-Z-infected midguts started from 28-h post inoculation (h p.i.) in low amounts, but in high amounts at late stages of infection. Immunofluorescence showed that NS2 ultimately concentrated on the nuclear membrane in BmN cells at late stages, indicating that NS2 might be associated with integral membrane protein.
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PMID:The non-structural protein NS-2 of Bombyx mori parvo-like virus is localized to the nuclear membrane. 2147 31

Porcine circovirus type 2 (PCV2) was retrospectively identified by serology in swine populations as an asymptomatic infection at least 25 years prior to the first reported case of PCV2-associated postweaning multisystemic wasting syndrome (PMWS). To investigate the sudden emergence of PMWS, viral sequences were amplified from frozen archived (1970-1971) porcine tissues and the complete genome of archival PCV2 was determined. The ORF1 gene product (viral DNA replicase) was homologous to contemporary PCV2 ORF1. In ORF2 (viral nucleocapsid gene) archival PCV2, a consistent linear nine-base sequence difference at base positions 1331 through 1339 was observed. The deduced amino acid sequence from these base changes alters the nucleocapsid conformation within the second immunogenic epitope from a hydrophobic (contemporary PCV2) to a hydrophilic (archival PCV2) configuration. To test the hypothesis that archival PCV2 was avirulent, cloned engineered archival and contemporary PCV2 genomes were constructed wherein the ORF1 gene was identical in each clone and the ORF2 gene (nucleocapsid protein) was sequence-identical in both clones except for the nine-base difference (bases 1331-1339), corresponding to archival and contemporary PCV2 viruses respectively. Clones were transfected into porcine kidney (PK) 15 cells and, after sequence confirmation, further passed in PK15 and 3D4/2 porcine alveolar macrophage cell cultures. Virulence trials in gnotobiotic piglets were conducted with cloned PCV2s. The data show that archival PCV2 is avirulent when compared to contemporary PCV2 and supports the hypothesis that the emergence of virulent contemporary PCV2 was a result of mutational events within this critical epitope after 1971.
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PMID:A nine-base nucleotide sequence in the porcine circovirus type 2 (PCV2) nucleocapsid gene determines viral replication and virulence. 2210 Mar 41

We expressed, purified, and characterized the helicase encoded by ORF1 of the Thermococcus nautili pTN2 plasmid (Soler et al. Nucl Acids Res 38, 5088-5104, 2010). The enzyme, which belongs to the SF1 family of helicases, possesses NTPase activity, with a strong preference for ATP and GTP as compared to CTP and TTP; dATP was also a substrate. Triphosphatase activity was strongly stimulated by single-stranded DNA and, to a lesser extent, by double-stranded DNA. Unwinding of duplexes comprising a fluorescent oligonucleotide was monitored by fluorescence polarization spectroscopy and by polyacrylamide gel electrophoresis. As observed for enzymes of the same family, pTN2 helicase displays a strong preference for duplexes comprising a 3' single-stranded extension and proceeds from the 3' to the 5' end of the loading strand. Under the conditions of the in vitro assay, pTN2 helicase did not appear to be recycled, but stayed bound to single-stranded DNA, which explains why high concentrations of enzyme are required to unwind long stretches of duplex DNA. The helicase enhances the synthesis of double-stranded DNA by pTN2 primase and by T. nautili PolB polymerase primed by pTN2 primase but it did not enhance synthesis by Taq DNA polymerase.
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PMID:The SF1 helicase encoded by the archaeal plasmid pTN2 of Thermococcus nautili. 2488 20


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