Cleavage of Adenovirus Type 2 DNA into Six Unique Fragments by Endonuclease R·RI

Pettersson, Ulf; Mulder, Carel; Delius, Hajo; Sharp, Phillip A.

doi:10.1073/pnas.70.1.200

Cited by 147 publications

(91 citation statements)

References 19 publications

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“…3a and Table 1). Based upon the length of the circular, doublestranded DNA, PM2, which has a molecular weight of 6.4 X 106 (19) and is present as an internal length standard, the maximal molecular weight of the 30-40S RNA in two different RNA preparations is 2.76 and 2.73 X 106 (Table 1). These data for the limit molecular weight are in excellent agreement with the molecular weight of 30-40S RNA that was determined under denaturing conditions (8,11) and by complexity analysis (20).…”

Section: Methodsmentioning

confidence: 99%

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Mangel

Delius

Duesberg

1974

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

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The structure and molecular weight of the 60-70S RNA complex and the 30-40S RNA species of Rous sarcoma virus were analyzed in an electron microscope after treatment of the RNAs with the bacteriophage T4 gene-32 protein to stretch out the RNA strands. Although all RNA preparations treated with gene-32 protein showed considerable heterogeneity in length, a significant fraction of the RNA retained its original sedimentation coefficient after treatment to allow the following conclusions to be made: The 30-40S RNA was confirmed to be a linear polynucleotide with a molecular weight of about 3 X 106. The 60-70S RNA exhibited a network structure with a molecular weight predominantly of about 6 X 106. Therefore, the subunit hypothesis for the 60-70S RNA is confirmed. A model for the structure and molecular weight of the 60-70S RNA postulates that the complex consists of two 30-40S RNA subunits held together at many points. This model elucidates the biological observation that the infectivity of RNA tumor viruses is proportional to the amount of 30-40S RNA in a virus preparation and not to the amount of 60-70S RNA.The structure and the molecular weight of tumor virus RNA have been difficult to study experimentally. The molecular weight is thought to range between a minimum of 3 X 106 and a maximum of 12 X 106 (1-10). This uncertainty is due mainly to the complex structure of the viral RNA (3) and to the difficulty in determining the exact content of RNA in purified virions (10). Extraction of the nucleic acid from virions followed by sedimentation reveals a major RNA species with a sedimentation coefficient of 60-70 S. When this RNA is placed in an environment wherein its secondary structure should be destroyed, the sedimentation coefficient decreases to 30-40 S and, at the same time, its electrophoretic mobility in sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels increases about 2-fold (11). The molecular weight of the 30-40S RNA species has been determined under denaturing conditions, and is about 3 X 106 (11). Thus, the 60-70S RNA was thought to be segmented, containing two, three, or four 30-40S RNA subunits held together by basepairing involving short complementary sequences.Although there is good, indirect, evidence for a subunit structure of the 60-70S RNA, nothing is known about how these subunits are linked. Linkage among 30-40S RNA subunits may be at only one site between two subunits, compatible with a relatively extended 60-70S RNA complex; or it may occur at several sites, compatible with a more compact, network-type 60-70S RNA complex. Furthermore, the available evidence does not exclude unequivocally that the conversion of tumor virus RNA from 60-70S to 30-40S may be due to an extreme conformational change rather than to a dissociation into subunits. Although there is no precedent for a conformational change that decreases the sedimentation coefficient but increases the electrophoretic mobility of nucleic acids, it was desirable to obtain independent evidence for the postulated subunit structu...

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Section: Methodsmentioning

confidence: 99%

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Mangel

Delius

Duesberg

1974

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

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“…All (13). Before use in hybridization experiments, the strands were degraded to an approximate size of 350 nucleotides by partial alkaline hydrolysis (11).…”

Section: Methodsmentioning

confidence: 99%

“…In the present report the relationship between nuclear and cytoplasmic RNA late after adenovirus infection has been studied by hybridization of RNA with separated strands of ad2 DNA and with separated strands of specific fragments which are obtained by cleavage of viral DNA with endonuclease EcoRI (11). This enzyme cleaves ad2 DNA (23 X 106 daltons) into six fragments, designated A-F according to decreasing size.…”

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confidence: 99%

Synthesis of Complementary RNA Sequences During Productive Adenovirus Infection

Pettersson

Philipson

1974

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

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Liquid RNA-DNA hybridization with separated strands of adenovirus type 2 DNA revealed that late nuclear RNA can hybridize to about 85% of the 1-strand and 10-15% of the h-strand, whereas late cytoplasmic RNA hybridizes to 65-70% and 25% of the 1-and h-strand, respectively. With separated strands from the six EcoRI fragments of adenovirus type 2 DNA as probes, it was shown that late nuclear RNA hydridizes to 85-90% of the 1-strand from all six EcoRI fragments. Since late cytoplasmic RNA hybridizes to 40-50% of the h-strand from both fragments EcoRI-B and EcoRI-C, complementary viral RNA sequences are synthesized during adenovirus infection. Complementarity between nuclear and cytoplasmic RNA could also be demonstrated by showing that late cytoplasmic RNA which had been preincubated with late nuclear RNA hybridized to a smaller fraction of the h-strand of fragment EcoRI-C than without preincubation. Double-stranded RNA which contains sequences that correspond to at least 60% of the viral genome was isolated from infected cells. However, less than 2% of the newly synthesized late RNA became double-stranded after incubation under annealing conditions, which suggests that RNA derived from one of the strands is present at a low concentration. Accordingly, it was shown that nearly all viral cytoplasmic RNA which is synthesized late after infection is derived from the 1-strand.

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“…Techniques presently used to isolate DNA from agarose gels are based on: (1) Electrophoresis either into a dialysis bag containing the agarose gel, or into a bag wrapped around the bottom of the gel holder [1,3,4]. Certain impurities still co-electrophorese with the DNA, while in addition often losses occur by sticking of the DNA to the dialysis bag [2].…”

Section: Introductionmentioning

confidence: 99%

An easy and efficient procedure for the isolation of pure DNA restriction fragments from agarose gels

Ledeboer

Hille

Schilperoort

1978

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein

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Cleavage of Adenovirus Type 2 DNA into Six Unique Fragments by Endonuclease R·RI

Cited by 147 publications

References 19 publications

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Synthesis of Complementary RNA Sequences During Productive Adenovirus Infection

An easy and efficient procedure for the isolation of pure DNA restriction fragments from agarose gels

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