AMV reverse transcriptase

The reverse transcriptase from the avian myeloblastosis virus (AMV RTase) is a prototype of avian retroviral RTases. The enzyme is a dimer (155 kDa) composed of nonidentical subunits a and /3. AMV RTase has multiple enzymatic activities including RNA-directed DNA polymerase (reverse transcriptase), DNA-directed DNA polymerase, RNase H, and DNA endonuclease. AMV RTase does not have 3 5 - or 5 3 -exonuclease activity. AMV RTase is a key reagent 

AMV RTase is stable under the reaction conditions and continuous polymerization occurs up to 1 hr. The yield of cDNA products is generally between 20 and 40%, depending on mRNA species. 

AMV RNase H is resistant to both hydrophobic and hydrophilic sulfhydryl reagents. NEM, a reagent specific to amino and SH groups, inactivates both polymerase and RNase H, but the RNase H activity is fourfold more resistant. The polymerase activity lost by DTNB or NEM modification can be partially (40-90%) recovered by incubation with 20 mM DTT (16). 

Pyrophosphate (Na-PPj) at a 0.5 mM concentration reduces the RTase activity to 50%. Among PP analogs, phosphonoformate (or foscarnet) is the strongest inhibitor to AMV RTase (8,20). The AMV RNase H activity is unaffected by the phosphonoformate. Na-PPj has also been reported to have no inhibitory effect on RNase H activity (21). The mode of RTase inhibition by phosphonoformate is noncompetitive (K = 5 to 100 pM) with respect to and depending on nucleotide substrates. The structurally related phosphonoacetate is not a RTase inhibitor. 

In some instances, however, PPj has been shown to block the degradation of RNA in RNA DNA hybrids and to impede the synthesis of anticomplementary DNA (22). Na-PPj ( 4 mM) has also been shown to increase the yield of full-length cDNAs significantly (9,23), making it a common practice to include PPj in the cDNA synthesis reaction with AMV RTase (but not with MoLV RTase). The simultaneous use of PP and actinomycin D results in an inhibition of the synthesis of both large and small cDNAs (4). 

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Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse transcriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the Superscript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically. 

The specific and irreversible binding of the platinated oligonucleotides on large molecular weight targets and in various media has been demonstrated in vitro by the arrest of AMV reverse transcriptase and protein synthesis and in HBL lOOrasl cells [73],... 

The 4 pi in each capillary are then used to dissolve 1 pi of dried down a-[32P]dATP (1 pCi/pl, specific activity 350 Ci/mmole). One microlitre of AMV reverse transcriptase (5 units/ ul) is diluted 1 5 in 1 x reverse transcriptase buffer, and 1 p of this diluted preparation added to each capillary and the contents mixed. 

Priming with p(dT)io-ia. AMV reverse transcriptase a - [32p]dATP dNTPs... 

Avian myeloblastosis virus (AMV) reverse transcriptase (AMV RT-XL 17 U///1) is purchased from Life Sciences Inc. (St. Petersburg, FL) the enzyme is aliquoted and stored at —20°... 

Maxam-Gilbert loading buffer 80% deionised formamide 10 mM NaOH 1 mM EDTA 0.02% xylene cyanol 0.02% bromophenol blue ddATP 2.0 mM ddTTP 2.0 mM ddCTP 0.66 mM ddGTP 1.33 mM dNTPs 2.5 mM AMV reverse transcriptase End-labelled primer... 

AMV reverse transcriptase (Life Science) tRNA carrier 5 mg/ml 5-end-labelled extension primer NaOAc 0.3 M (pH 6.0)... 

AMV Reverse transcriptase isolated from avian myeloblastosis virus. This enzyme catalyzes the polymerization of nucleotides and characterized by RNA-dependent DNA polymerase, DNA-dependent DNA polymerase activity besides the RNase H activity but lacks the 3 -5 exonuclease activity. The RNase H activity can cause the degradation of the RNA strand of an RNA DNA duplex, which is a disadvantage that can limit the complete synthesis of the total cDNA. The AMV Reverse Transcriptase is suitable for reverse transcription of fragments containing secondary structure due to its high optimum temperature (42 °C) for its activity. 

Chapter 36 describes a protocol for an isothermal chain reaction that makes use of the presence of DNAprimere, T7 promoters, T7 polymerase, RNaseH, and AMV reverse transcriptase to produce large quantities of nucleic acid when the primers used match an RNA (or DNA) template. This system has the advantage that no thermal cycling block is required, and hence the number of samples that can be processed daily is not limited by the availability of specialized equipment. 

AMV reverse transcriptase (RT Seikagaku America, Rockville, MD, see Note 5) Use as supplied store at -20°C. Alternatively, Bst polymerase (Bio-Rad, Richmond, CA) can be used for DNA priming. 

Add z pL template (RNA analyte or PI primed DNA) to bring the total volume to 25 pL. Template volume will vary depending on concentration and desired number of input molecules, usually lO molecules see Note 10). For negative control reactions template volume will equal 0. (Summary of standard reaction conditions 40 mAf Tris-HCl, pH 8.5, 50 mAf KCl, 12 mAf MgClj, 2 mAf NTPs (each), 1 mAf dNTPs (each), 10 mAf DTT, 0.2 pAf PI, 0.2 pAf P2,15% DMSO, 100 pg/mL BSA, 20 U T7 RNA Polymerase, 8 U AMV Reverse Transcriptase, 0.2 U RNase H, 12.5 U RNAguard , 10 molecules of template). 

The probe for active genes was prepared by extracting total RNA from asynchronous, logarithmically growing HeLa cells, purifying the polyadenylated RNA by poly-U Sephadex chromatography, and reverse transcribing with AMV reverse transcriptase in the presence of [ P]-labeled dCTP. 

Alternatively, 0.5 pi (4-5 U) AMV reverse transcriptase from Promega (Cat No. M5101) and 0.5 pi (2 U) of Taq DNA polymerase from Boehringer Mannheim (Expand high fidelity PCR system, Cat No. 1732641) can be mixed and added to the reaction mixture. 

The precise reaction conditions are flexible. The protocol described here is based on that supplied with the Superscript II reverse transcriptase In earlier studies (14) using 15 U AMV reverse transcriptase (Promega, Southampton, UK) we used 2 5-fold lower concentrations of dNTPs and one-third the amount of oligo d(T) primer, in reactions containing 2.5 U RNasin and 5 mM MgCl2 together with reaction buffer supplied with the enzyme Incubation conditions for this enzyme were 15 mm at 42°C. 

Total RNA from white adipose tissues was purified with DNase I and used for RT-PCR. cDNA synthesis with AMV Reverse Transcriptase XL. cDNA fragments were amplified with a thermal cycler for initial denaturation at 94 °C for 90s, followed by 18-34 cycles of amplification each cycle consisted of 94 °C for 30s, 60-63 °C for 30s, 72 °C for 45s. Acidic ribosomal phosphoprotein PO (36B4) 18S was used as the internal standard. After agarose electrophoresis, the results were quantified using Scion Image (Scion Corporation, Frederick, MD, US) and normalized to 36B4 mRNA expression. PCR primers were as follows ... 

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