Moloney murine leukemia virus reverse transcriptase

Georgiadis MM, lessen SM, Ogata CM, Telesnitsky A, Goff SP, Hendrickson WA. Mechanistic implications from the structure of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase. Structure 1995 3 879-892. 

Tanese N, Goff SP. Domain structure of the Moloney murine leukemia virus reverse transcriptase mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci USA 1988 85 1777-1781. 

Moloney murine leukemia virus reverse transcriptase. 

A commercial viral enzyme preparation of Moloney murine leukemia virus reverse transcriptase (MoMuLV) having Poly(A)dT (primer for RT) was used. 50 pi of the MoMuLV preparation was combined with a mixture of dATP, dGTP, dCTP and [3H]dTTP nucleotides. 

Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) at 200 U/pL (Life Technologies). 

Erkel, G. Anke, T. Velten, R. Steglich, W. Antibiotics from Basidiomycetes. XXXIX. Podoscyphic Acid, A New Inhibitor of Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus Reverse Transcriptase from a Podoscypha Species. Biosci. 1991, 46, 442-450. 

The detection of HIV-related proteins is one of the most challenging tasks. This is especially true because AIDS should be diagnosed as early as possible to enable an early and effective therapy of this infection. Pavski and Le (57) used the aptamer strategy to detect reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1). A direct and specific ACE method was proposed using laser-induced fluorescence (ACE/LIF) as detection principle. Single-stranded DNA aptamers as probes fluorescently labeled were synthesized. The resulting aptamer is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-l RT was stable, with calibration curves linear up to 50 nM (6 /xg/mL) HIV-1 RT concentration. Both... 

Reverse transcriptase activity. Water extract of Zingiber officinale (Rh), Bupleurum falcatum, Scutellaria baicalensis (Rt), Pinellia ternata (Tu), Zizyphus jujuba (Fr), Panax gim seng (Rt), and Glycyrrhiza glabra (Rh), at a concentration of 200 pg/mL, was active on Moloney murine leukemia virus and HlVzo2 L... 

Tanese NT, Telesnitsky A, Goff SP. Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function. J Virol 1991 65 4387-4397. 

Das, D., and Georgiadis, M. M. (2004). The crystal structure of the monomeric reverse transcriptase from Moloney murine leukemia virus. Structure 12, 819-829. 

Selected naturally occurring flavonoids have been shown to inhibit three reverse transcriptases (RT) (avian myeloblastosis RT, Rous-associated virus-2 RT, and Moloney murine leukemia virus, or MMLV, RT) when poly(rA)oligo(dT) 12-18 or rabbit globin mRNA was used as a template. Amentoflavone, scutellarein, and quercetin were the most active compounds, and their effect was concentration dependent. The enzymes exhibited differential sensitivity to the inhibitory effects of the flavonoids. These flavonoids also inhibited rabbit globin mRNA-directed MMLV RT-catalyzed... 

In 1994, six novel drimane-type sesquiterpenoids, mniopetals A-F (1-6) (Fig. (1)), were isolated by Anke and Steglich from the fermentation broth of Canadian Mniopetalum sp. 87256 [1, 2]. These natural products are known to inhibit the enzymatic activity of RNA-directed DNA-polymerases (reverse transcriptases) of the human immunodeficiency virus (HIV)-l, avian myeloblastosis virus (AMV), and moloney murine leukemia virus (MMuLV) as shown in Table 1. In addition, mniopetals exhibit moderate antimicrobial and cytotoxic activities. In the following year, a structurally and biologically similar natural product kuehnero-... 

Although many enzymes that are active in the processing of nucleic acids, such as nucleases, DNA polymerases, or reverse transcriptases, have acidic residues in the active sites and require divalent cations for activity, such cations have been repotted only for a few published structures. One published structure of reverse transcriptase from Moloney murine leukemia virus, (MMLV RT) shows a single metal bound in the active site (15), whereas none of the available structures of HIV-1 RT show bound metals. In addition, the structures of MMLV RT and E. coli RNase H with bound metals have been solved at a lower resolution than the... 

Reverse transcription of RNA is sometimes used, e.g., for clone selection by colony hybridization (Verbeek and Tijssen, 1988). Although both avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV) reverse transcriptase can be used, we prefer the AMV enzyme if there are no strong secondary structures in the RNA (MMLV enzyme can be used at higher temperatures, even up to 55°C). The optimal pH range for the AMV enzyme is very narrow (should be 8.3) whereas the MMLV enzyme is active in a range of 7.6-8.3. Superscript reverse transcriptase from BRL is a cloned MMLV enzyme from which the RNase H activity has been deleted. 

Mature mRNA transcripts (sense strand) from eukaryotic cells can be purified and then reverse transcribed, with the assistance of a reverse transcriptase enzyme (from Moloney murine leukemia virus, MMLV), into complementary DNAs (cDNAs) that will anneal with the mRNA transcripts by Watson-Crick base pairing to give anti-parallel DNA/RNA duplexes or double helices. The poly(A) tail in each mature mRNA transcript is actually a usefiil handle for each reverse transcriptase reaction. Thereafter, DNA/RNA duplexes must be broken down with the assistance ofRNAse enzymes (specific for the hydrolysis of RNA phospho diester links) and a sense strand of DNA constructed instead on each cDNA single strand so that equivalent, more stable antiparallel DNA/DNA duplexes are generated instead, with the assistance of a DNA polymerase enzyme. In this instance, the poly(T) tail in each cDNA molecule turns out to be important for the DNA polymerase reaction ... 

M-MulV Reverse transcriptase isolated from moloney murine leukemia virus. This enzyme is also characterized by RNA-dependent DNA polymerase, DNA-dependent DNA polymerase and weak RN ase H activities but lacks the 3 -5 exonuclease activity. M -MulV reverse transcriptase is able to synthesize full-length cDNA fragments from longmRNA.Thetemperaturefortheoptimalenzymeactivity is 37 °C. Becauseofthe weak RNase H activities of the M-MulV reverse transcriptase, it is more suitable for the synthesis of long cDNA fragments. 

Reverse transcriptase (RT). Moloney murine leukemia virus (M-MLV) RT (200 U/ j,L) should be purchased from Gibco-BRL The enzyme is supplied with a vial of 5X reaction buffer (250 mM Tris-HCl, pH 8 3, 375 mM with KCl, and 15 mM MgCl2) and a vial of 100 mM DTT. It is advisable to aliquot the enzyme into 3-4 smaller portions and use them in succession Store at -20°C RNasin (20-40 U/p.L), recombinant or purified from human placenta, should be purchased from Promega (Madison, WI)... 

Cells were washed with ice-cold PBS and total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction (27). 5 pg total RNA was reverse-transcribed (RT) into cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase and oligo (dT)i primer by incubating the reaction mixture (15 pL) at 37 C for 90 min. The polymerase chain reaction (PCR) was performed as described previously (22). A final volume of 25 pL contained dNTPs (each at 200 pM), IX reaction buffer, 0.4 pM each primer (iNOS forward ... 

The reverse transcriptase (RTase) from the Moloney murine leukemia virus (MoLV) possesses both DNA polymerase and RNase (H and D) activities. It is monomeric (80 kDa) in solution but is presumed to function as a dimer. The ratio of the polymerase to RNase H activities and many of MoLV RTase functions are similar to those of AMV RTase. In contrast to AMV RTase, however, MoLV RTase does not possess DNA endonuclease activity. The gene for MoLV RTase has been cloned and expressed in . colt, and both wild-type RTase (RNase H" ) and modified RTase (RNase H ) are commercially available. MoLV RTase is thus a reliable reagent which shares with AMV RTase much of the role of cDNA synthesis in molecular cloning. Modified MoLV RTase has proven to be particularly useful in the synthesis of full-length cDNAs. 

Finally, due to the above conqretitive mechanism, quercetin, quercetagetin, m)uicetin, and especially baicalein were potent inhibitors of murine leukemia virus (MLV) (Rauscher and Moloney strains) and HTV-l reverse transcriptases (Ono et al. 1989). Comparative studies with other flavonoids (hydroxyflavones, dihydroxyflavones and polyhydroxyflavones, and flavanones) revealed that the presence of both the unsaturated double bond between po-... 

C, H2604, Mr 282.38, yellowish solid, mp. 105 C. An unsaturated dioxocarboxylic acid from cultures of a Tasmanian Podoscypha species (Basidiomycetes). P. inhibits reverse transcriptases of viruses of the avian myeloblastosis and Moloney murine leukemia types. Lit Z. Naturforsch. C 46,442 (1991). - [CAS 135863-69-31... 

---