RNA as a template with E. Coli DNA polymerase

Cavalieri, Liebe F.; Carroll, Elizabeth

doi:10.1016/0006-291x(70)90192-0

Cited by 30 publications

(6 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We report here that E. coli DNA polymerase I is able to make complementary copies from a variety of natural heteropolymeric RNAs, thus confirming the results of Loeb et al (12). We also show that a high ratio of enzyme to template RNA molecules is required to elicit RNA-dependent DNA polymerase activity from the E. coli polymerase, and that the unbalanced incorporation of thymidine observed by others (7,10,13) can be eliminated.…”

supporting

confidence: 77%

See 1 more Smart Citation

Conditions for Using DNA Polymerase I as an RNA-Dependent DNA Polymerase

Gulati

Kacian

Spiegelman

1974

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments. The molar ratio of enzyme to template is critical; below a certain level, synthesis is not observed. Hybrids formed with the complementary DNA are of comparable specificity and stability to those formed with complementary DNAs synthesized by viral RNA-directed DNA polymerase. Synthesis of dA-dT polymers, a common occurrence with this enzyme, can be eliminated by including distamycin in the reaction mixture.RNA-directed DNA polymerase (1) from avian myeloblastosis virus (AMV) (2) lacks template specificity (3), making it useful for synthesizing highly labeled DNA complements from a wide variety of RNA species. These complementary DNAs (cDNAs) have been used as hybridization probes, affording an extremely useful and sensitive technique for detecting and quantitating nucleic acid sequences (4-6).Only AMV is a practical source for the enzyme, and its relative unavailability has prevented many from using these techniques for resolving a variety of interesting problems. Previous attempts (7-11) to use E. coli DNA polymerase for copying RNA led to indifferent results until the recent reported success by Loeb and his colleagues (12). Because of the potential usefulness of the enzyme to those interested in preparing cDNA hybridization probes, we felt that further investigation of the E. coli enzyme was warranted. We report here that E. coli DNA polymerase I is able to make complementary copies from a variety of natural heteropolymeric RNAs, thus confirming the results of Loeb et al. (12). We also show that a high ratio of enzyme to template RNA molecules is required to elicit RNA-dependent DNA polymerase activity from the E. coli polymerase, and that the unbalanced incorporation of thymidine observed by others (7, 10, 13) can be eliminated. MATERIALS AND METHODSDNA Polymerase. Three different batches of E. coli polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) were used for these experiments.Two preparations were kindly provided by Dr. Lawrence Loeb (Institute for Cancer Research, Philadephia) and were prepared according to Slater et al. (14 Hybridization reactions were carried out in (a) 50% formamide, 0.4 M NaCl, 50 mM EDTA, 0.1% sodium dodecyl sulfate at 370 or (b) 0.3 M NaCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate, 20 mM sodium phosphate (pH 7.0) at 680. The annealing reactions were analyzed for hybrid formation as follows: (a) Cesium sulfate equilibrium centrifugation was performed as described (4). (b) Thermal elution with hydroxylapatite was performed in 0.12 M sodium phosphate (pH 6.7). The hybridization reaction was diluted 100-fold with 0.12 M sodium phosphate and loaded onto a 2-ml bed volume hydroxylapatite column at 60°. The column was then washed with 14 ml of 0.12 M sodium phosphate buffer. The temperature was raised in small increments, and the column was eluted with 6 m...

show abstract

supporting

confidence: 77%

“…Previous attempts (7)(8)(9)(10)(11) to use E. coli DNA polymerase for copying RNA led to indifferent results until the recent reported success by Loeb and his colleagues (12). Because of the potential usefulness of the enzyme to those interested in preparing cDNA hybridization probes, we felt that further investigation of the E. coli enzyme was warranted.…”

mentioning

confidence: 99%

Conditions for Using DNA Polymerase I as an RNA-Dependent DNA Polymerase

Gulati

Kacian

Spiegelman

1974

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…coli DNA polymerase I is about as versatile in the alternative use of RNA and DNA as primers for nucleotide addition in vitro, as is the avian myeloblastosis virus enzyme [lo] which exemplifies the so-called "reverse transcriptases". Other evidence [4][5][6]9,11] leads to a qualitatively similar conclusion with regard to the alternative use of RNA and DNA as templates for nucleotide selection. While not detracting from the probable importance of RNA-directed DNA synthesis in the life cycle of RNA tumour viruses, such findings emphasise the inadvisability of assuming that "reverse transcription" behaviour is diagnostic for tumourvirus enzymes-a point already made by others [6, Irrespectivc of its biological significance, our interest in DNA extension of RNA chains in vitro has been based on its possible use as a method for the study of untranscribed DNA sequences, which might include termination signals for transcription.…”

Section: Discussionmentioning

confidence: 56%

Extension of RNA Molecules with DNA An

Peters

Hayward

1972

European Journal of Biochemistry

View full text Add to dashboard Cite

Sequences in DNA, which act as signals for the termination of transcription, may lie wholly or partially beyond the distal end of the DNA region actually transcribed into RNA. In a hybrid formed between an intact RNA molecule and its DNA complement, any such untranscribed signal should lie in the region of single-stranded DNA close to the 3'-hydroxyl terminus of the RNA. We show here that hybrids of this type, formed on circular bacteriophage DNA, act as efficient primers for Escherichia coli DNA polymerase I and T4-induced DNA polymerase, under the normal conditions for :DNA synthesis in vitro. The DNA product is covalently linked to the 3'-end of the RNA, and co-purifies with it in CsCl equilibrium centrifugation. Priming by RNA is readily detectable even when DNA primers are also present. These and other features of the reaction suggest that it might form the basis of a general method for the study of DNA sequences beyond the termini of RNA chains.For the controlled expression of genetic information, the transcription of RNA from DNA must be initiated and terminated a t defined loci. There is evidence that RNA chain extension ceases when a termination signal encoded in the DNA is reached and recognised by the RNA-polymerising enzyme [i]. I n some, but not all cases this recognition seems to require the co-operation of accessory proteins, such as rho-factor in Escherichia coli [a].To understand the mechanism of RNA termination, it seems desirable to study the base sequence of termination signals. If these signals are fully transcribed from the DNA into the distal, 3'-ends of the RNA molecules involved, it should be comparatively easy to discover their nature through sequencing Enzymes. RNA polymerase or nucleoside triphosphate : RNA nucleotidyltransferase (EC 2.7.7.6) ; DNA polymerase or deoxynucleoside triphosphate : DNA nucleotidyltransferase (EC 2.7.7.7) ; pancreatic deoxyribonuclease or deoxyribonucleate oligonucleotide hydrolase (EC 3.1.4.5) ; ribonuclease T, or ribonucleate guaninenucleotide-2'-transferase (EC 2.7.7.26) ; alkaline phosphat,ase or orthophosphoric monoester phosphohydrolase (EC 3.1.3.1). studies on the 3'-regions of appropriate RNA species [3].However, it is quite conceivable that termination signals lie wholly or partially beyond the distal end of that portion of the DNA which is actually transscribed. Novel techniques would then be necessary for the study of terminator sequences. The present work was motivated by the desire to develop such a technique .If one imagines an intact RNA molecule hybridised with the DNA strand to which it is complementary, the presumptive untranscribed termination signal should lie in the region of single-stranded DNA following the 3'-hydroxyl terminus of the RNA, and presumably not far beyond it. A DNA polymerase capable of using this RNA terminus as a primer would synthesise a polydeoxyribonucleotide, the initial portion of which should be complementary to the sequence of interest on the DNA template strand. The sequence of the synthetic polynucleotide could...

show abstract

“…First, poly[d(A-T)] is a "preferred" template for each. In addition, the E. coli enzyme has been shown to catalyze an RNA-dependent reaction with either natural RNA (22) or certain synthetic RNAs (23) as templates. Thus, the findings reported here suggest that poly(U) or poly(G) might serve as a rapid and simple means of differentiating cellular polymerases from the viral enzyme in crude enzyme preparations.…”

Section: Discussionmentioning

confidence: 99%

“…5 show clearly that the base composition of the polyribonucleotide does play an important part in determining the strength of the inhibition. The apparent dissociation constants (Ki's) for the four polyribonucleotides tested ranged from 0.2 (micromolar concentration of polynucleotide phosphorus) for poly(U) to 22 for polyC). Expressed in terms of pg/ml, the K a's were 0.08 for poly(U), 0.25 for poly(G), 4.4 for poly(A), and 9.7 for poly(C) Single-stranded polyribonucleotides do not serve as templates for polymerization by the DNA polymerases of RNA tumor viruses under the usual reaction conditions (5).…”

Section: Dna Polymerase Preparationsmentioning

confidence: 99%

Inhibition of the DNA Polymerase of Rauscher Leukemia Virus by Single-Stranded Polyribonucleotides

Tuominen

Kenney

1971

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The DNA polymerase of Rauscher murine leukemia virus is strongly and specifically inhibited by nontemplate, single-stranded polyribonucleotides with either the resident viral RNA, native calf-thymus DNA, or poly[d(A-T)l as templates. These inhibitory homo-polymers are apparently bound to the template site of the polymerase, since they interact competitively with the template. The strength of the inhibition depends on the particular homopolymer used: poly(U) > poly(G) >> poly(A) > poly(C). The Ki for poly(U) was 0.08 Mg/ml, which represents an apparent affinity six times greater than that observed for viral RNA. No such inhibition was observed with a highly purified DNA polymerase from mouse embryos or the Escherichia coli enzyme.There are now many reports of RNA-dependent DNA polymerases in oncogenic RNA viruses (1-8). Although similar activities have been detected in partially purified preparations from normal (9, 10) and leukemic (11-13) cells, a satisfactory assay for the specific determination of these enzymes in cell extracts has not been described. The required specificity can only be achieved when properties that are unique to the viral enzymes are clearly understood. The work described here concerns one possible unique property.The published findings of Spiegelman et al. (5) show that synthetic, single-stranded homopolyribonucleotides do not serve as templates for the DNA polymerase of the avian myeloblastosis virus. The present communication describes the effects of these homopolymers on the polymerization of deoxyribonucleotides by the enzyme of the Rauscher murine leukemia virus. The enzyme-polynucleotide interactions described here may provide a basis for a specific assay for viral polymerases. MATERIALS AND METHODS Virus purificationRauscher murine leukemia virus (RLV) was purified from two different sources. One source was a 10-fold mouse (strain CFW-S) plasma concentrate (lot number RPV-HL-68-4 of Hazleton Laboratories) that contained 5.48 log units (spleen enlarging)/ml. The viruses from this source were purified according to published methods (3). Rauscher virus from JLS-V5 tissue culture fluids was also purified by this procedure after an initial sedimentation at 78,000 X g. DNA polymerase preparationsCrude Rauscher virus DNA polymerase was prepared by detergent lysis of purified RLV at 0-4°C for 10 min in a solution containing 0.05 M Tris . HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 8 mM MgCl2, 4 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, and 150-300 ,ug of viral protein per ml.DNA polymerase was repeatedly purified from 16-to 18-day-old BALB/c mouse embryos by a method similar to that described by Alberts et al. (14) for the purification of DNA polymerase I from Escherichia coli. ln the procedure, decapitated embryos were homogenized at 4°C in a pH 8.1 buffer containing 5 M NaCl-0.05 M MgCl2-5 mM 2-mercaptoethanol-0.02 M Tris HCl. A 105,000 X g supernatant was prepared from the homogenate after it had been subjected to sonication (Bronwill Biosonik with a 12-mm diameter probe at maximum o...

show abstract

RNA as a template with E. Coli DNA polymerase

Cited by 30 publications

References 8 publications

Conditions for Using DNA Polymerase I as an RNA-Dependent DNA Polymerase

Conditions for Using DNA Polymerase I as an RNA-Dependent DNA Polymerase

Extension of RNA Molecules with DNA An

Inhibition of the DNA Polymerase of Rauscher Leukemia Virus by Single-Stranded Polyribonucleotides

Contact Info

Product

Resources

About