• BMB Reports
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• v.31 no.6
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• pp.585-589
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• 1998

The herpes simplex virus type-1 (HSV-1)-encoded DNA polymerase consists of two subunits, the products of the UL30 and UL42 genes. UL30 and UL42 were coexpressed in Sf9 cells infected with recombinant baculoviruses carrying the two genes. The UL30 and UL42 gene products remained tightly associated throughout the purification, which led to a near homogeneous heterodimer composed of the DNA polymerase and UL42 protein. The DNA polymerase-UL42 protein heterodimer, purified from the recombinant baculovirus-infected Sf9 cells, showed the same high degree of processivity of deoxynucleotide polymerization as the enzyme purified from the HSV-1 infected primate cells. Like the latter, it contained a 3'-5' exonuclease activity that specifically hydrolyzes an incorrectly matched nucleotide at the 3' terminus of a primer, thereby contributing to the fidelity of DNA replication.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.2
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• pp.209-218
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• 1995

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, has been implicated in T7 DNA replication, recombination, and repair. Purified gene 2.5 protein has been shown to interact with the phage encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phage that contain null mutants of gene 2.5 were constructed by homologous recombination. These mutant phage $(T7\Delta2.5)$ cannot grow in Escherichia coli. After infection of E. coli with $T7\Delta2.5$, host DNA synthesis is shut off, and $T7\Delta2.5$ DNA synthesis is reduced to less than $1\%$ of wild-type phage DNA synthesis (Kim and Richardson, 1993, Proc. Natl. Aca. Sci. USA, 90, 10173-10177). A truncated gene 2.5 protein $(GP2.5-\Delta21C)$ deleted the 21 carboxyl terminal amino acids was constructed by in vitro mutagenesis. $GP2.5-\Delta21C$ cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than $1\%$ of the DNA synthesis observed in wild-type phage-infected cells. $GP2.5-\Delta21C$ has been purified to apparent homogeneity from cells overexpressing its cloned gene. Purified $GP2.5-\Delta21C$ does not physically into「act with T1 gene 4 protein as measured by affinity chromatography and immunoblot analysis. The mutant protein cannot stimulate T7 gene 4 protein activity on RNA-primed DNA synthesis and primer synthesis. These results suggest that C-terminal domain of gene 2.5 protein is essential for protein-protein interactions.

• Journal of Plant Biology
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• v.37 no.1
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• pp.77-83
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• 1994

DNA uptake in dry embryos of rice by DNA imbibition was detected by monitoring the expression of chimeric vectors. The selective markers of expression vectors used were ${\beta}-glucuronidase$ ronidase (GUS) and hygromycin phosphotransferase (HPT) genes under the control of CaMV35 S promoter. Frequency of transient expression of the foreign gene was generally 30-50% varying according to the types of vectors and rice cultivars. Dot blot analysis and DNA sequence analysis of inverse polymerase chain reaction products showed that selected rice in hygromycin B (HmB) medium had HPT gene and CaMV35S promoter DNA sequence in genomic DNA of rice. To investigate what ratio of rice having two marker genes simultaneously as rice embryos imbibed the vector DNA having two HPT and GUS gene, transform ants selected in lImB medium were subjected to PCR for GUS gene. It was shown that about 90 percentage of surviving ones in HmB medium had GUS gene.S gene.

• Journal of Plant Biology
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• v.38 no.2
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• pp.129-136
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• 1995

Complementary DNA of the genomic RNA of odontoglossum ringspot virus Cymbidium strain (ORSV-Cy) was synthesized from polyadenylated viral RNA and cloned. Selected clones containing the viral RNA-dependent RNA polymerase gene of the virus has been sequenced by automated sequencing system. The complete nucleotide sequence of an open reading frame is 1377 base pairs in length, and encodes a protein of 458 amino acids about 52, 334 D. The 52 kD protein of ORSV shares four sequence motifs characteristic of viral RNA-dependent RNA polymerase. Comparison of the ORSV 52 kD protein sequence with that of other five viruses in tobamovirus group showed 76.0 to 60.7% homologies at the amino acid level and the conservation of the four motifs betwen the viruses.

• BMB Reports
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• v.32 no.5
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• pp.492-496
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• 1999

We have investigated the biochemical properties of the herpes simplex virus type 1 (HSV-1) DNA polymerase without the UL42 protein (Pol), purified from insect cells infected with a recombinant baculovirus containing the UL30 gene. BSA and DTT have inhibitory effects on dAMP incorporation. Pol showed a greater turnover rate of steady-state single nucleotide incorporation at 12 mM $MgCl_2$ than at 2 mM $MgCl_2$. However, it showed a greater processivity of DNA synthesis at lower $MgCl_2$ concentration (1 mM, 2 mM) than at a higher $MgCl_2$ concentration (12.5 mM). These results are consistent with a slow DNA dissociation at lower $MgCl_2$ concentrations. Pol does not incorporate a correct nucleotide into the primer with an incorrect nucleotide at the end; instead, it preferentially excises the incorrect nucleotide at the 3' end of the primer. Pol has DNA polymerase activity at pHs 6.5 and 7.5 but little at pHs 5.5, 8.5, and 9.5. It has exonuclease activity at pHs 6.5, 7.5, and 8.5 but little at pHs 4.5, 5.5, and 9.5. The finding that Pol has exonuclease activity but not DNA polymerase at pH 8.5 suggests that DNA binds to Pol, but deoxynucleotide binding or incorporation does not occur at pH 8.5.

• Journal of Veterinary Clinics
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• v.15 no.1
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• pp.140-145
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• 1998

The present study was carried out for the rapid and accurate detection of Anaplasma marginale in cattle using Polymerase Chain Reaction. One pair of primer, BAP-2 and AL34S, were designed to amplify a 409 Up fragment of the A marginale membrane surface protein encoding beta($msp{\beta}l$) gene with a hilly sensitive and specific PCR. A marginale isolated from naturally infected calf in Chonbuk area were used to obtain target genomic DNA for PCR. This study showed that a 409 bp of $msp{\beta}l$ gene fragment could be detected as little as 15 fg of purified A marginale genomic DNA. The amplified fragment with PCR was checked for the identification of $msp{\beta}l$ gene by enzyme restriction and sequencing. Also, the target DNA extracted directly from blood were used in the PCR reactions without prior purification to shorten the detection time. The PCR in the present study was considered convenient and rapid method for the detection of A marginale in whole blood of infected cattle.

• Korean Journal Plant Pathology
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• v.10 no.3
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• pp.157-162
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• 1994

Erwinia rhapontici causes soft-rot disease in a number of plants such as onion, garlic and hyacinth. There has been no report that E. rhapontici produces pectate lyase. Pel gene was cloned from genomic DNA of the parasitic soft-rot E. rhapontici polymerase chain reaction by using synthetic oligonulceotide primers designed from the pel 1 to E. carotovora. The recombinant plasmid pJE101 containing pectate lyase gene, when introduced into E. coli DH5$\alpha$, produced pectate lyase an macerated hyacinth tissue.

• The Korean Journal of Food And Nutrition
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• v.13 no.1
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• pp.1-5
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• 2000

Detection for heat-labile enterotoxin(LT) of enterotoxigenic Escherichia coli(ETEC, EC81, O148:H28) and enterotoxin of enterotoxigentic Clostridium perfringents type A(CP, NCTC8238, Hobbs serotype 2) by use of a polymerase chain reaction (PCR) assay were positive reaction, which using LT gene-specific primers of ETEC with a detection limit equivalent from 100ng/${\mu}\ell$ to 1 pg of a DNA fragment of 417-bp in EC81 and enterotoxin gene-specific primers of CP with a detection limit equivalent from 100ng/${\mu}\ell$ to 10pg of a DNA fragment of 364-bp in NCTC8238. Detection for a LT gene of ETEC highly appeared 10-fold sensitivity than an enterotoxin gene of CP.

• International Journal of Oral Biology
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• v.46 no.3
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• pp.140-145
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• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• Korean Journal of Veterinary Research
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• v.38 no.3
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• pp.565-573
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• 1998

To study the specific tools for the diagnosis of Newcastle disease virus (NDV) in chicken, polymerase chain reaction (PCR) and its presumable conditions were evaluated for the detection of hemagglutinin-neuraminidase (HN) gene of NDV RNA. For these purposes, Kyojeongwon strain of the NDV was propagated in allantoic cavity of SPF embryonating chicken eggs, and viral RNA was extracted from fractionated virus after the allantoic fluids were ultracentrifuged with sucrose gradient. The first-strand cDNA was then made for the HN gene of NDV RNA by reverse transcription at $42^{\circ}C$ for 1 hour using specific primer complementary to the HN gene. The single-stranded cDNA was used as template in the PCR of the HN-DNA, and various conditions of the PCR were evaluated to set up method for the specific detection of the HN-DNA. The PCR conditions promising for the detection of HN gene consist of preheating at $94^{\circ}C$, 5 min, 30 cycles of denaturation at $94^{\circ}C$, 1 min, annealing at $55^{\circ}C$, 1 min and polymerization at $72^{\circ}C$, 2 min, and a cycle of extension at $72^{\circ}C$, 5 min. when NDVs of allantoic fluids without fractionation were applied to the above PCR condition, the HN genes were detected effectively not only from Kyojeongwon but from other velogenic strains such as Herts and a field isolate.