Burkitt''s Tumors Contain Particles Encapsulating RNA-Instructed DNA Polymerase and High Molecular Weight Virus-Related RNA

Burkitt's lymphomas, linked by previous studies with the DNA-containing Epstein-Barr virus, contain an RNA related in sequence to that of Rauscher leukemia virus. The present study establishes that the viral-related RNA found in Burkitt's tumors is a 70S component encapsulated with RNA-instructed DNA polymerase in a particle possessing a density characteristic of RNA tumor viruses. Further, the DNA synthesized by the Burkitt particles hybridizes specifically to the RNA of Rauscher leukemia virus. Thus, four features characteristic of a known oncogenic RNA agent are also exhibited by particles found with a high (87%) frequency in Burkitt's tumors. The relation between the RNA particle and the Epstein-Barr virus and their etiological roles remains to be elucidated. However, relevant to these issues is the finding reported here that the presence of Epstein-Barr virus information in nonneoplastic cells does not lead to the production of the RNA particles that have been detected now in three different human neoplasias, including leukemias, breast cancer, and Burkitt's disease.     

Particles Containing RNA-Instructed DNA Polymerase and Virus-Related RNA in Human Breast Cancers

Human breast cancers contain an RNA related to that of mouse mammary tumor virus. In 79% of the breast malignancies examined, this type of RNA is a 70S-component encapsulated with RNA-instructed DNA polymerase in a particle possessing the density characteristics of RNA tumor viruses. Further, the DNA synthesized by the human RNA enzyme complex hybridizes specifically with the RNA of mouse mammary tumor virus. Thus, four features diagnostic of agents similar to mouse mammary tumor virus are also exhibited by a particle found with high frequency in human breast cancers. The accumulating evidence for the involvement of RNA tumor viruses in at least some human neoplasias is becoming increasingly compelling.     

Particles with RNA of High Molecular Weight and RNA-Directed DNA Polymerase in Human Brain Tumors

We have previously shown that neoplastic cells of human breast cancers, leukemias, lymphomas, and sarcomas contain particles similar to the viruses that have been established as etiologic agents of these diseases in mice. The present paper concerns tumors of the central nervous system for which no suitable animal model or corresponding virus exists. Nevertheless, using the simultaneous detection test, we showed that human brain tumors contain 70S RNA and RNA-directed DNA polymerase encapsulated in a particulate component possessing a density of 1.17 g/ml. These particles satisfy the three diagnostic criteria that characterize RNA tumor viruses of animals. 24 Out of 26 (92%) of the most malignant (glioblastoma and medulloblastoma) brain tumors examined contained these virus-like entities.     

Evidence of Particle-Associated RNA-Directed DNA Polymerase and High Molecular Weight RNA in Human Gastrointestinal and Lung Malignancies

Previous communications have demonstrated that neoplastic cells of human breast cancers, leukemias, lymphomas, sarcomas, and brain tumors contain particles with similar diagnostic attributes as those found in RNA oncornaviruses. The present paper concerns malignancies of the gastrointestinal and pulmonary systems for which, like brain tumors, no suitable animal model or corresponding virus exists. By means of the simultaneous detection assay, these tumors have been found to contain 70S RNA and RNA-directed DNA polymerase encapsulated in particulate components possessing densities of 1.16-1.17 g/ml. Twelve out of 17 (70%) colon carcinomas, three out of five (60%) gastric carcinomas, all of three rectal carcinomas, and seven out of ten (70%) lung carcinomas contained detectable levels of these virus-like entities. None of the corresponding normal tissues was positive.     

Accessibility of DNA in Chromatin to DNA Polymerase and RNA Polymerase

The accessibility of DNA in chromatin to both exogenous DNA polymerase and RNA polymerase is slight when compared to isolated DNA. DNA in extracted chromatin is somewhat more accessible to these enzymes than is DNA in the chromatin of isolated nuclei; and the DNA template of chromatin is more accessible to DNA polymerase than to RNA polymerase. In these experiments we have given much attention to the technique of scintillation counting, since artifacts arising in this procedure can lead to erroneous conclusions.     

DNA Polymerase Activity Associated with RNA Tumor Viruses

C-type RNA viruses, originating in mouse, cat, hamster, and viper, catalyze the synthesis of DNA from its constituent deoxyribonucleoside triphosphates. Both the rate and extent of the reaction were significantly enhanced by brief treatment of the intact virus with ether. A proportion of the newly synthesized DNA was associated with virions when intact virus was used as template; this was not the case with ether-treated virus. In both cases, DNA extracted from the reaction mixture sedimented slowly, at about 2-4 S.     

Multiple Molecular Species of Escherichia coli DNA Polymerase

DNA polymerase activity from Escherichia coli can be demonstrated in various sized molecules ranging in molecular weight from about 10,000 to 120,000 or higher. The characterization of the smaller species is difficult because of their pronounced tendency toward aggregation; the smallest apparently aggregates most readily. The results indicate the following molecular weight classes: 10,000-20,000; 40,000-50,000; 75,000-85,000, and 100,000-120,000. The same classes were obtained with several methods of analysis of material that had been purified in a number of ways, one of which is a new DNA-acrylamide gel chromatographic procedure. The lowest molecular weight species shows no exonucleolytic activity. A proteolytic inhibitor, phenylmethyl sulfonylfluoride, did not eliminate the small active molecules, although proteolysis of high molecular weight DNA polymerase (109,000) has been shown by others to produce fragments of about 75,000 molecular weight. Either there is a naturally occurring polymerase protein of about 20,000 molecular weight, capable of aggregation with itself and with certain other molecules (e.g., exonucleases), or there are certain bonds in a large, native polymerase molecule that are especially susceptible to proteolysis without destroying activity.     

RNA Polymerases of Maize. Purification and Molecular Structure of DNA-dependent RNA Polymerase II

Nuclear DNA-dependent RNA polymerase II has been purified from leaves of Zea mays by a new procedure that improves enzyme stability and thus permits more manipulation during purification. The purification procedure includes a heating step, gel filtration on Sepharose 6B and 4B, and chromatography on DEAE- and DNA-celluloses. This method of purification yields an enzyme that exhibits maximal activity when denatured DNA is used as a template. Electrophoresis of highly purified enzyme on polyacrylamide gels containing sodium dodecyl sulfate indicates that maize RNA polymerase IIa is composed of several polypeptide subunits. The most highly purified preparations contain polypeptides with molecular weights of 200,000, 160,000, 35,000, 25,000, 20,000, and 17,000.     

The 3′-Terminal Nucleosides of the High Molecular Weight RNA of C-Type Viruses

Tumor virus RNAs from several mammalian and one reptilian species were purified; their 3'-terminal nucleoside was identified by separation of the trialcohols produced by periodate oxidation followed by reduction and tritiation with NaB(3)H(4). Each virus contained uridine as the predominant terminal nucleoside. Molecular weight estimations based on the tritiation reactions were consistent with a structure consisting of four subunits.     

Different Molecular Weight Chitosan-Based Membranes for Tissue Regeneration

Natural polymers, such as chitosan, obtained from chitin, are been widely studied for use in the tissue regeneration field. This study established a protocol to attain membranes made from this biopolymer, consisting of high or low molecular weight chitosan. The biocompatibility of these membranes was histologically evaluated, comparing them to collagen membrane surgically implanted in rat subcutaneous tissue. Fifteen Holtzmann rats were divided in three experimental groups: High and Low Molecular Weight Chitosan membranes (HMWC and LMWC) and Collagen membranes (C—control group); each of them with three experimental periods: 7, 15 and 30 days. As a result, after the seven days evaluation, the membranes were present and associated with a variable degree of inflammation, and after the 15 and 30 days evaluations, the membranes were absent in all groups. It is concluded that the chitosan-based membranes were successfully attained and presented comparable resorption times to collagen membranes.     

DNA Replication in Escherichia coli: Location of Recently Incorporated Thymidine Withi Molecules of High Molecular Weight DNA

More than 50% of the [(3)H]thymidine incorporated during short pulses into the DNA of Escherichia coli 15T(-) can be extracted by alkali as high molecular weight DNA. Density gradient centrifugation and digestion with exonuclease I suggest that these large pieces of DNA are composed of newly synthesized DNA attached to pre-existing material at the 3' end of the molecule.     

DNA聚合酶链反应检测石蜡包埋人脑组织中弓形虫的研究

孕妇感染弓形虫是胎儿先天性畸形的危险因素。本文运用DNA聚合酶链反应检测30例先天性畸形儿尸检石蜡包埋脑组织中弓形虫。结果表明,30例先天性畸形儿石蜡脑组织切片经体外扩增后出现阳性条带者13例,而对照组无1例阳性(P<0.05)。研究结果进一步支持弓形虫感染与胎儿先天性畸形有密切关系。     

Comparative Properties of RNA and DNA Templates for the DNA Polymerase of Rous Sarcoma Virus

Several natural RNAs were compared with respect to their template activities for the DNA polymerase of Rous Sarcoma Virus during a 2-hr incubation period. 60-70S viral RNA was found to be a 5- to 10-fold better template than heat-dissociated Rous viral RNA, influenza virus RNA, tobacco mosaic virus RNA, or ribosomal RNA. Denatured salmon DNA is a little better, and poly(dAT) is 2-4 times better as a template for the enzyme than is 60-70S Rous viral RNA. The 60-70S RNAs of different strains of avian tumor viruses have very similar template activities for a given avian tumor virus DNA polymerase. Oligo(dT) or oligo(dC) were found to enhance the template activity of heat-dissociated Rous viral RNA 20- to 30-fold, and that of other natural RNAs tested one- to several-fold. DNA syntheses of 1-24% were obtained during a 2-hour incubation of the enzyme with the above RNA templates. The results suggest that the enzyme prefers partially doublestranded or hybrid regions of RNAs for optimal DNA synthesis, but certain regions of single-stranded RNA can also serve as templates.Poly(dAT) competes with viral RNA for purified DNA polymerase during DNA synthesis, as would be expected if RNA- and DNA-dependent DNA synthesis was performed by at least one common active site of the same enzyme.     

Detection of Leishmania siamensis DNA in Saliva by Polymerase Chain Reaction

Polymerase chain reaction was used to detect Leishmania siamensis DNA from clinical samples collected from six leishmaniasis patients during 2011–2012. The samples used in this study came from bone marrow, blood, buffy coat, saliva, urine, and tissue biopsy specimens. Saliva was a good source for L. siamensis DNA by polymerase chain reaction. L. siamensis DNA was also found in saliva of an asymptomatic case-patient. Levels of L. siamensis DNA in saliva decreased until being undetectable after treatment. These levels could be used as a marker to evaluate efficacy of the treatment. A larger study is needed to evaluate this method as a screening and survey tool to study the silent background of Leishmania infection among the at-risk population.Leishmaniasis is a neglected tropical diseases caused by an obligate intracellular protozoa belonging to the genus Leishmania. The disease is transmitted to vertebrate hosts by infected female sand flies taking a blood meal.¹ Leishmaniasis presents in three clinical forms; visceral, cutaneous, and mucocutaneous.² Clinical presentation of leishmaniasis depends on the species of Leishmania and the immunity of the host.³Detection and species identification of the parasites is essential for prognostic and therapeutic reasons and surveys.⁴ Several laboratory techniques have been used for diagnosis of Leishmania infection. They are microscopy, culture, immunologic techniques (enzyme-linked immunosorbent assay, direct agglutination test, and recombinant protein K39 dipstick test), and molecular techniques (polymerase chain reaction [PCR] and quantitative PCR).^5–7 In comparisons of microscopic examination, culture, and PCR in detecting Leishmania parasites, PCR has shown to have a significantly higher sensitivity than culture and microscopic examination (97%, 78%, and 76%^8–10 sensitivity, respectively). New cases of leishmaniasis caused by L. siamensis, a novel species of Leishmania, have been documented in patients in Thailand^11–17 and Myanmar (unpublished data). The infection was described in immunocompromised patients, mostly persons infected with human immunodeficiency virus (HIV).^11–17 Clinical presentations of these patients have included visceral,^11–13 diffuse cutaneous,¹⁵ and overlapping diffuse cutaneous and visceral forms.¹⁴With low prevalence of leishmaniasis in Thailand and Myanmar, screening tests for leishmaniasis such as enzyme-linked immunosorbent assay, direct agglutination test, and recombinant protein K39 dipstick test are not readily available. Moreover, sensitivity and specificity of these serologic tests for detection of L. siamensis infection have never been fully documented. Diagnosis of L. siamensis infection relies on microscopic examination, culture, and detection of parasite DNA by PCR.^11–15 Although microscopic examination and culture of Leishmania parasites have high specificity, they require experience and have low sensitivity. The PCR is commonly used to diagnose leishmaniasis caused by L. siamensis. Bone marrow, blood, buffy coat, tissue, saliva, and urine have been successfully used for detection of L. siamensis DNA by PCR.¹⁴Saliva has shown to be a good source for L. siamensis DNA.¹⁴ There are also reports of using saliva to identify other Leishmania species.^18–20 Collection of saliva is noninvasive and convenient for field studies. We describe a PCR to amplify the internal transcribed spacer1 (ITS1) gene of L. siamensis from six infected patients and compare it with specimens collected from patients and different clinical presentations. Details of clinical presentations and management of some patients enrolled in this study have been described.¹⁴Bone marrow, blood or saliva was smeared on glass slides, and fixed with absolute methanol (Sigma-Aldrich, St. Louis, MO) for one minute. The slides were then stained with Giemsa (Sigma-Aldrich) in phosphate buffer, pH 7.2. Tissue biopsy specimens were stained with hematoxylin and eosin. Stained slides were then examined under a light microscope (Olympus, Tokyo, Japan).Schneider''s insect medium (Sigma-Aldrich) containing 20% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Sigma-Aldrich) was used for culturing Leishmania parasites. One hundred microliters of bone marrow, blood, or saliva was inoculated into 5 mL of culture media in a 25-cm³ flask and incubated at 25 ± 2°C. Cultures were inspected for parasites every 24 hours by using an inverted microscope (Olympus).Two hundred microliters of blood or bone marrow and 50 μL of buffy coat were used for DNA extraction by using a blood DNA extraction kit (Invisorb^® Spin Blood Mini Kit (STRATEC Molecular, Berlin, Germany). Thirty milliliters of urine or 1 mL of saliva were centrifuged for 5 minutes at 5,000 × g, and the pellets were collected and used for further DNA extraction. Tissue specimens, urine pellets, and saliva pellets were used for DNA extraction by tissue DNA extraction (Invisorb^® Spin Tissue Mini Kit) according to the manufacturer''s instructions. Extracted DNA was eluted in 80 μL of elution buffer. Quantity and quality of the extracted DNA was determined by using a Nanodrop 2000c Apparatus (Thermo Scientific, Singapore). Extracted DNA samples were kept at −80°C for long-term storage. Blood, saliva, and urine were collected from three healthy uninfected persons and used for the PCR as negative controls.The PCR was performed in a final volume of 25 μL containing approximately 100 ng of extracted DNA, 10 μM of each primer, 25 mM of MgCl₂, 2 mM of dNTPs, and 1 unit of Taq DNA polymerase (Fermentas, Pittsburgh, PA). The primers were designed to anneal specifically to the ITS1 regions of ribosomal RNA (rRNA) of Leishmania parasites described by Spanakos and others:²¹ (LeF: 5′-TCC GCC CGA AAG TTC ACC GAT A-3′ and LeR: 5′-CCA AGT CAT CCA TCG CGA CAC G-3′). The PCRs were performed in a PCR Mastercycler^® Pro (Eppendorf, Hamburg, Germany) with conditions as follows: denaturation at 94°C for 4 minutes; followed by 40 cycles at 94°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. Aliquots of the PCR amplicons were analyzed by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized with Quantity One Quantification Analysis Software version 4.5.2 (Gel Doc EQ System; Bio-Rad, Hercules, CA). The extracted DNA samples from an uninfected persons and a no DNA template (double-distilled water) were used as negative controls in every PCR. Reactions in which either or both negative controls contained bands were discarded.Confirmatory testing for PCR was performed by using another set of primers to amplify the partial small subunit (SSU) rRNA genes of the Leishmania parasite. The primer sequences (R221: 5′-GGT TCC TTT CCT GAT TTA CG-3′ and R332: 5′-GGC CGG TAA AGG CCG AAT AG-3′) and PCR conditions were described by Van Eys and others.²²The PCR amplicons were ligated into the pGEM-T Easy Vector (Promega, Madison, WI). Ligation reactions mixture was composed of 5 μL of 2× Rapid ligation buffer, 3 μL of PCR products, 1 μL pGEM-T Easy Vector, and 1 μL double-distilled water. The ligated vectors were transformed into DH5α competent cells and chimeric plasmids were screened by blue–white colony selection system. The suspected positive colonies were cultured and used for further plasmid DNA extraction by using the Invisorb^® Spin Plasmid Mini Kit following the manufacturer''s instructions. Purified plasmids were sequenced by 1st BASE DNA sequencing services (1st Base Laboratories, Kuala Lumpur, Malaysia) by using universal forward T7 primer. Nucleotide sequences were analyzed by using BioEdit Sequence Alignment Editor Version 7.0.9.0 (www.mbio.ncsu.edu/bioedit/bioedit.html), and consensus sequences were compared with available sequence data in a GenBank by BLAST search (www.ncbi.nlm.gov/BLAST).The study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (IRB no. 385/55).Patient 1 was a 46-year-old Thai man who was a rubber planter from southern Thailand. He has been given a diagnosis with HIV infection in 2003 and received boosted lopinavir and lamivudine. His CD4+ T cell count was 175 cells/mm³, and serum virus levels were < 40 copies/mL. He also had Evans syndrome (an autoimmune disorder with destruction of erythrocytes, platelets, and leukocytes), a left knee ulcer, and hepatosplenomegaly. A bone marrow study showed Leishmania amastigotes within macrophages. Bone marrow, blood, discharge from the ulcer, saliva, and urine were cultured and DNA was extracted for PCR.He improved after two weeks of intravenous amphotericin B deoxycholate (1 mg/kg/day), followed by 400 mg of itraconazole/day. A recurrence after two months of itraconazole therapy was re-treated with three weeks of intravenous liposomal amphotericin B (3 mg/kg/day), followed by 400 mg of itraconazole per day. Blood and saliva were collected for PCR three months after re-treatment and were negative. Details of this patient were reported by Chusri and others.¹⁴Patient 2 was 30-year-old pet store owner from southern Thailand who had been given a diagnosis of HIV infection in 1999. The patient received tenofovir, lamivudine, and nevirapine. His CD4+ T-cell count was 111 cells/mm³ and viral RNA was not detectable (< 40 copies/mL). He had multiple papules and plaques with ulceration and discharges. He also had anemia, thrombocytopenia, and hepatosplenomegaly. Bone marrow, papule, and ulcer biopsy specimens showed Leishmania amastigotes within macrophages. Bone marrow, blood, tissue biopsy specimens, and saliva and urine samples were used for culture and PCR. The patient received intravenous amphotericin B deoxycholate (1 mg/kg/day) for two weeks, followed by 400 mg of itraconazole per day. Blood and saliva were collected three months later and were negative for Leishmania and no recurrence was observed. This patient was reported by Chusri and others.¹⁴Patient 3 was a 60-year-old man with diabetes who lived in Yangon, Myanmar and had not previously traveled abroad. He showed development of fever, multiple infiltrative skin lesions, and oral ulcers. A skin biopsy specimen indicated Sweet''s syndrome or acute febrile neutrophilic dermatosis, which is a skin disease with fever and painful skin lesions that is commonly present on arms, neck, face, and back. He was treated with systemic corticosteroids for two months without improvement. He came to Thailand for further evaluation. Multiple erythematous, shiny, infiltrative plaques; nodules on face, trunk, and extremities; and oral ulcers and white patches on the buccal mucosa were observed. Lymph nodes, liver, and spleen were not palpable. Complete blood counts, blood urea nitrogen levels, creatinine levels, and liver function test results were within normal limits. Results for antinuclear antibodies, antibodies against HIV, and C-reactive protein levels were unremarkable.A new skin biopsy specimen of a trunk nodule showed diffuse histiocytic infiltrate and multinucleated giant cells in the upper and deep dermis. Many round-to-oval small organisms were present in histiocytes and fibrous stroma. They stained positive with Periodic Acid–Schiff (PAS) stain. Numerous small yeast-liked organisms were present in histiocytes and in the stroma. Some of these organisms were large and contained small basophilic dots in cytoplasm near nuclei stained positively with PAS stain but not with Gomori''s methenamine silver stain and Giemsa. This finding suggested the presence of Leishmania parasites. Blood samples, tissue biopsy specimens, saliva and urine samples were used for culture and were negative. PCR testing of the ITS1 region of the rRNA gene in blood, skin biopsy specimen, urine, and saliva, and DNA sequencing identified L. siamensis. The patient was treated with intravenous amphotericin B for 40 days (total dose = 2.1 grams). After regression of the cutaneous lesions, he was discharged. When seen again two months later, he had gained weight but a few skin lesions were still present. He returned home and was lost to follow-up.Patient 4 was the 22-year-old daughter of patient 3; she came to Thailand with her father. She was healthy and lived with her father in Yangon. Results of her physical examination were unremarkable. Saliva and urine samples were collected and used for culture; these samples were negative for L. siamensis. PCR testing of the ITS1 region of the rRNA gene in saliva DNA, followed by DNA sequencing, identified L. siamensis. Whole blood and buffy coat were then collected for Leishmania detection by culture, staining with Giemsa, and PCR. Only PCR identified L. siamensis in buffy coat. We planned to follow-up the patient for detection of Leishmania parasites without treatment of leishmaniasis but the patient was lost to follow-up.Patient 5 was a 45-year-old Thai man living in Chiang Rai, Thailand. He has been given a diagnosis of HIV infection in 2005. He had a CD4+ T cell count of < 50 cells/mm³. He was treated with stavudine, lamivudine, and nevirapine. In 2007, He showed development of lumpy skin lesions that were first not investigated. He was later hospitalized with fever, oral candidiasis, pancytopenia, pancreatitis, type 2 diabetes mellitus, epistaxis, perianal abscess, urinary retention, and abnormal liver function test results. Skin biopsy specimens from lesions present for more than five years showed epidermal hyperplasia, diffuse fibrosis, dilated blood vessels, and mild perivascular lymphohistiocytic infiltrates in the dermis. There were a few clumps of small parasites in histiocytes and extracellularly between collagen fibers in the upper dermis. They stained with Giemsa. Blood, tissue biopsy specimen, and saliva and urine samples were used for culture but were negative. A PCR of ITS1 region of the rRNA gene in blood, tissue biopsy specimen, and saliva DNA, followed by DNA sequencing, confirmed the presence of L. siamensis. The patient was then confirmed as having cutaneous leishmaniasis. He was severely debilitated and died of systemic bacterial infection without treatment of leishmaniasis.Patient 6 was a 34-year-old Burmese man from Yangon who was seropositive for HIV for six years. He was treated with truvada, legalon, isoniazid, rifampicin, ethambutal, and pyrazinamide. Five months later, he was hospitalized with high fever and diarrhea. He was empirically treated with clarithromycin and moxifloxacin. He showed development of multiple, umbilicated, erythematous papules on his neck, arms, and chest wall. Skin biopsy specimens showed a moderately dense superficial and deep perivascular and interstitial histiocytic infiltrate. Many small yeast-like organisms were present in the cytoplasm of histiocytes and between collagen fibers. They stained with PAS and Giemsa, leading to diagnosis of cutaneous leishmaniasis. Blood, tissue, saliva, and urine samples were cultured. A PCR of the ITS1 region of the rRNA gene in blood, skin biopsy specimen, and saliva DNA, followed by DNA sequencing, identified L. siamensis. The patient was treated with liposomal amphotericin B and anti-tuberculosis therapy was continued. Clinical recovery resulted within one month.Specimens were obtained from patients with different clinical presentations of leishmaniasis (

A New Synthetic RNA-Dependent DNA Polymerase from Human Tissue Culture Cells

Two DNA polymerases that can copy synthetic RNA polymers are present in human tissue culture cells. These enzymes which have each been purified about 500-fold, are present in both HeLa cells, which are derived from a cervical carcinoma, and in WI-38 cells, a normal diploid strain originating from human embryonic lung tissue. These synthetic RNA-dependent DNA polymerases are identified by their ability to copy efficiently the ribo strand of synthetic oligonucleotide-homopolymer complexes, and differ in this respect from the known DNA-dependent DNA polymerases found in HeLa cells. The template requirements of these new DNA polymerases resemble that of the RNA-dependent DNA polymerases of the RNA tumor-viruses.     

聚合酶链反应检测致病性钩端螺旋体DNA的研究

以黄疸出血型ＲＧＡ菌株对所有致病性钩端螺旋体特异的ＤＮＡ克隆片段序列为基础合成聚合酶链反应引物Ｇ１Ｇ２。用该对引物扩增各群型致病性钩端螺旋体微量ＤＮＡ，均获阳性结果，对非致病性钩端螺旋体和其他细菌不扩增。纯化的钩端螺旋体ＤＮＡ５ｐｇ经聚合酶链反应后，琼脂糖凝胶电泳可以目测。用地高辛配基标记的特异性探针可检测到５ｆｇ即相当于１条钩端螺旋体ＤＮＡ量的扩增产物。将该对引物扩增早期钩体病患者血清标本，阳性率为８２％，显著高于ＭＡＴ和ｄｏｔ－ＥＬＩＳＡ阳性率。由此表明，ＰＣＲ技术是灵敏、特异、快速的钩端螺旋体病早期诊断方法，还可用于流行病学调查。     

The 3′-Terminal Nucleosides of the High Molecular Weight RNA of Avian Myeloblastosis Virus

The RNA isolated from avian myeloblastosis virus was fractionated by sucrose density gradient centrifugation. The 3'-OH terminal nucleosides of various fractions were determined by periodate oxidation followed by tritiated borohydride reduction. The 60-70S fraction and the 35S RNA derived from it by heating both have adenosine as the major terminal nucleoside, with cytidine as the next most frequent terminal. Control samples of tRNA(met) (f. coli) and 28S ribosomal RNA from mouse ascites tumor cells gave the expected terminal residues and molecular weights.     

逆转录聚合酶链反应检测蚊体内黄病毒的研究

本文报告了应用自行设计的黄病毒通用引物和逆转录聚合酶键反应成功地扩增了实验室脑内接种感染白纹伊蚊体内登革病毒RNA和海南省非流行区野外捕获的白纹伊蚊和骚扰阿蚊体内黄病毒RNA。该法灵敏度高,特异性强,获得结果迅速。能检出5只蚊虫中含有1只阳性蚊的混合标本中的登革病毒RNA。整个过程只要数小时就可完成。这对蚊体内黄病毒的快速检测及流行病学调查、防治监测等研究,具有重要意义。     

Primer Requirement and Template Specificity of the DNA Polymerase of RNA Tumor Viruses

Polyribonucleotides will act as efficient templates for the DNA polymerases found in the virions of avian myeloblastosis virus and mouse leukemia virus if a short complementary oligodeoxyribonucleotide primer is added. Synthesis of the complementary polydeoxyribonucleotide continues until an amount of polymer equal to the amount of initial template has been produced. The two viruses show slightly different specificities toward the four homoribopolymers. Polydeoxyribonucleotides are generally much poorer templates than the homologous polyribonucleotides, in most cases yielding no detectable synthesis. The DNA polymerase of RNA tumor viruses, therefore, have the same requirements for activity as do other DNA polymerases, except that they prefer polyribonucleotides over polydeoxyribonucleotides as templates.