No abstract available.
Antiretroviral Therapy, Highly Active* ; Humans ; RNA-Directed DNA Polymerase*

No abstract available.
Genes, pol* ; HIV-1 ; Humans* ; RNA-Directed DNA Polymerase ; Zidovudine*

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.
Biotechnology ; Chimera ; DNA ; DNA, Single-Stranded* ; Genome, Bacterial* ; Retroelements ; Reverse Transcription ; RNA ; RNA-Directed DNA Polymerase

Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA onto chromosomal ends to compensate for sequence loss during replication. It has been detected in a variety of human malignancies, suggesting that such activity may play a role in the tumorigenic process. To determine whether telomerase is reactivated in malignant fibrous histiocytoma, 12 tissue samples with this tumor were analyzed for the telomerase activity by a radioactive PCR-based TRAP (telomeric repeat amplification protocol) assay. All of the tumors were further investigated for the expression of human telomerase RNA (hTR) by an in situ hybridization (ISH). Telomerase activity was detected in one (8.3%) sample. Expression of hTR was demonstrated in 7 (58.3%): one telomerase-positive and six telomerase-negatives. These data indicate that the reactivation of telomerase is an uncommon event and not an important factor involved in tumorigenesis in malignant fibrous histiocytoma. It is noteworthy that 50% of the patients with grade 2 tumors expressed hTR, suggesting that telomerase RNA may be useful as a marker for identifying tumor aggressiveness earlier than the conventional histopathologic grading scale.
Carcinogenesis ; DNA ; Histiocytoma, Malignant Fibrous* ; Humans ; In Situ Hybridization ; RNA* ; RNA-Directed DNA Polymerase ; Telomerase*

OBJECTIVETo synthesize highly pure HBV post-transcriptional regulatory element (HPRE) via transcription in vitro by T7 RNA polymerase.

METHODSHPRE gene was amplified by PCR from a template containing HBV complete genomic DNA and cloned into plasmid pGEM-11zf. The cloned DNA sequence was transcribed by T7 RNA polymerase.

RESULTSThe construction of HPRE gene recombinant plasmid and production of HPRE via transcription in vitro was successful.

CONCLUSIONIn vitro transcription by T7 RNA polymerase can be used to synthesize highly pure HPRE.

DNA-Directed DNA Polymerase ; DNA-Directed RNA Polymerases ; Hepatitis B virus ; genetics ; RNA Processing, Post-Transcriptional ; RNA Splicing ; RNA-Binding Proteins ; physiology ; Transcription, Genetic ; Viral Proteins

To investigate resistance to lamivudine (3TC), we examined the incidence of M184V in 20 HIV-1 patients treated with 3TC for 13.1 +/- 9 months. Fourteen of 20 patients had been exposed to zidovudine (ZDV) or didanosine (ddl) prior to 3TC therapy. Nested PCR targeting to reverse transcriptase (RT) and direct sequencing were performed for peripheral blood mononuclear cells sampled serially. There were resistance mutations to ZDV in at least 9 patients at baseline, although there was no resistance mutation to 3TC. We could detect M184V in 6 (30%) out of 20 patients. The incidence of M184V increased as the duration of therapy prolongs (13% in samples<12 months; 47% in samples gtoreq 12 months). The frequency of mutation M184V was higher in patients with previous mutation to ZDV than in patients with wild type. Resistance mutation was not detected in 7 patients. This study shows that resistance to 3TC tends to develop rapidly in patients with baseline mutations or two drugs combination therapy than in those treated simultaneously with triple drugs. This report is the first on resistance to 3TC in Korean AIDS patients.
Didanosine ; HIV-1* ; Humans ; Incidence ; Lamivudine* ; Polymerase Chain Reaction ; RNA-Directed DNA Polymerase ; Zidovudine

BACKGROUND: Real-time reverse transcriptase PCR (rRT-PCR) is widely used to detect novel influenza A/H1N1. We had observed several cases with positive result for influenza A and negative result for novel influenza A/H1N1 during a novel influenza A/H1N1 outbreak. The causes of those results were investigated in this study. METHODS: A total of 913 cases tested with rRT-PCR for novel influenza A/H1N1 (Real-time Ready Influenza A/H1N1 Detection Set, Roche Diagnostics, Germany) during 25 August 2009 to 8 September 2009 was enrolled in this study. Cases showing positive result for influenza A (M gene) and negative result for novel influenza A/H1N1 (H1 gene) were tested with multiplex RT-PCR for seasonal influenza and novel influenza A/H1N1 (Seeplex FluA ACE Subtyping kit, Seegene, Korea), and the amplicons were directly sequenced. RESULTS: One hundred and eleven cases (12.2%) were positive for novel influenza A/H1N1. Twenty-seven cases (3.0%) were positive for influenza A, but negative for novel influenza A/H1N1. Subtypes of influenza A were determined in 25 cases by multiplex RT-PCR and nucleotides sequencing. One novel influenza A/H1N1, six seasonal influenza A/H1N1, three seasonal influenza A/H3N2, and 15 influenza A/H9N2 were detected. CONCLUSIONS: Subtypes of influenza A were determined in most cases with positive result for influenza A and negative result for novel influenza A/H1N1. Several cases with seasonal influenza A were detected. Even if a nonepidemic period of seasonal influenza, tests for seasonal influenza A can help in the differential diagnosis of influenza.
Diagnosis, Differential ; Influenza, Human ; Nucleotides ; Reverse Transcriptase Polymerase Chain Reaction ; RNA-Directed DNA Polymerase ; Seasons

No abstract available.
Chin* ; Humans ; Membranes* ; Prostate* ; Prostatic Neoplasms* ; RNA, Messenger* ; RNA-Directed DNA Polymerase*

PURPOSE: Sarcomas have rarely been analyzed for telomerase, which is an RNA-dependent DNA polymerase to maintain telomeres and prevent telomere shortening. This study was undertaken to determine telomerase activity and the expression of the telomerase subunits human telomerase RNA (hTR) and telomerase reverse transcriptase (TERT) in soft tissue sarcomas. MATERIALS AND METHODS: Twenty three sarcomas were analyzed for the telomerase activity by a radioactive PCR-based TRAP assay. All of the samples were further investigated for the expression of hTR by in situ hybridization and for TERT and p53 by immunohistochemistry. RESULTS: Telomerase activity was detected in four (17%) samples. Expression of hTR was demonstrated in 11 (48%) cases, whereas TERT was expressed in 20 (87%).Of the four telomerase-positive tumors, three were positive for both hTR and TERT, and one was positive only for TERT. p53 overexpression was observed in nine (39%) tumors. The frequency of p53 expression increased as the tumor grade advanced (p= .064). CONCLUSION: These data indicate that the reactivation of telomerase is an uncommon event in human soft tissue sarcomas. The high frequency of the expression of hTR and TERT in these tumors suggests that telomerase activity may be regulated at the transcriptional level and an additional event leading to telomerase activation exist.
Humans* ; Immunohistochemistry ; In Situ Hybridization ; RNA ; RNA-Directed DNA Polymerase ; Sarcoma* ; Telomerase* ; Telomere ; Telomere Shortening

The purpose of this study was to develop species-specific real-time quantitative PCR (RT-qPCR) primers for use in the detection of Aggregatibacter actinomycetemcomitans. These primers were designed based on the nucleotide sequences of the RNA polymerase beta-subunit gene (rpoB). We assessed the specificity of the primers against nine strains of A. actinomycetemcomitans, eight strains (three species) of the Haemophilus genus, and 40 strains of 40 other oral bacterial species. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC 33384T. Our data reveal that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 2 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these qRT-PCR primers are suitable for application in epidemiological studies.
Base Sequence ; Cinnarizine ; DNA ; DNA-Directed RNA Polymerases ; Haemophilus ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity