A Nuclease from Neurospora Crassa Conidia Specific for Single-Stranded Nucleic Acids

Rabin, E. Z.; Preiss, Benjamin; Fraser, Murray

doi:10.1080/00327487108081946

Cited by 40 publications

(22 citation statements)

References 25 publications

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“…(control no. 21-1-758) by the technique of Rabin, Preiss, and Fraser (20). For each enzyme isolation, 250 g of frozen conidia paste was thawed overnight at 0-4'C and suspended in 800 ml of 0.05 M glycylglycine buffer, pH 7.0.…”

Section: Dna Preparation Human Epidermoid Cancer Cells (Kb-mentioning

confidence: 99%

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Deoxyribonucleic acid strandedness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum.

Samaha¹,

Irvin²

1975

J. Clin. Invest.

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A B S T R A C T This study shows that tritiated thymidine labeled DNA prepared from mammalian cells by the Marmur technique is a pure preparation of nucleic acid that is composed essentially of two populations of molecules. One molecular population consists of primarily double-stranded nucleic acid, while the other population is of double-stranded nucleic acid with significant single-stranded regions. The double-stranded DNA with single-stranded regions can, depending upon the length of the single strand, behave as "native" DNA or "denatured" DNA on methylated albumin kieselguhr (MAK) column chromatography. Using MAK chromatography we have separated the DNA into a saltelutable fraction composed of primarily double-stranded molecules and an alkaline-elutable fraction containing double-stranded nucleic acid with variable length, singlestranded regions. Endonuclease enzyme removal of the single-stranded regions from the alkaline fraction DNA yields nucleic acid that behaves identically to the salt elutable DNA. Exonuclease removal of the singlestranded regions suggests they are located primarily at the ends of the molecules. Our data show that the alkaline-elutable DNA differs from salt-elutable DNA only in that the former has significant single-stranded regions.Sera of patients with systemic lupus erythematosus (SLE) selected for anti-DNA by hemagglutination bind significantly less to the alkaline fraction DNA than the salt fraction DNA. This difference in binding clearly does not represent simply an affinity for double-stranded vs. single-stranded nucleic acid since the alkaline fraction DNA contains predominately double-stranded nu-

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Section: Dna Preparation Human Epidermoid Cancer Cells (Kb-mentioning

confidence: 99%

“…Y.). Our isolation procedure was modified according to suggestions of the Rabin et al (20) (26). The capacity of MAK columns is about 1 mg of DNA for 10 ml of MAK suspension (16).…”

Section: Dna Preparation Human Epidermoid Cancer Cells (Kb-mentioning

confidence: 99%

Deoxyribonucleic acid strandedness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum.

Samaha¹,

Irvin²

1975

J. Clin. Invest.

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“…The compounds used for inhibition included hydralazine HCl, hydralazines-RSA, hydralazine25-HSA and metabolitev5-RSA. Anti To exclude the possibility that the antibodies being detected by the "native" DNA-binding assay were not to the small areas of single-strandedness known to exist in commercial DNA (18,19) but to the double-stranded structure of native DNA, native DNA (Worthington Biochemical Corp.) was digested with an exonuclease isolated from Neurospora crassa conidia (Miles Laboratories, Inc.), an enzyme which has been demonstrated to be specific for single-stranded DNA (prepared by heat) and which does not attack DNA in the native form (20,21 ANA were determined by using normal rabbit peripheral white cells as a substrate with a fluoresceinated goat antirabbit IgG antiserum at a concentration of 3 mg/ml. The antiserum was prepared and labeled in this laboratory.…”

Section: Methodsmentioning

confidence: 99%

Induction of antibodies to nuclear antigens in rabbits by immunization with hydralazine-human serum albumin conjugates.

Yamauchi¹,

Litwin²,

Adams³

et al. 1975

J. Clin. Invest.

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“…The reaction was stopped either by the addition of EDTA or by heating the sample to 650 for 5 min to inactivate the enzyme (9). Neurospora crassa single-strand specific exonuclease (provided by K. Bartok) was prepared as described by Rabin et al (10); the purified enzyme preparation contained 120 units/mi. One unit of nuclease activity is defined as that amount of enzyme which causes the release Electron Microscopy.…”

Section: Methodsmentioning

confidence: 99%

Arrangement of sequences in the inverted terminal repetition of adenovirus 18 DNA.

Garon¹,

Berry²,

Rose³

1975

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

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In contrast to the single-stranded circular molecules produced with denatured DNA from other adenoviruses, there was associated with nearly all circular molecules of adenovirus type 18 a visible, duplex projection.These projections had a mean contour length of 0.31 ± 0.12 ,sm, equivalent to approximately 3% of genome length. Individual (3)(4)(5). At present the DNA of only one other group of viruses, the adenovirus-associated viruses (AAV), has been found to contain a similar type of repetition (6). Because of these repetitious sequences, the ends of single-stranded DNA molecules can self-anneal to form circles closed by a duplex hydrogenbonded segment. Although the arrangement of sequences within the repetition should be apparent from the morphology of the closure region, in the case of adenovirus DNA this region apparently has been too short to visualize by electron microscopy. We have recently found, however, that preparations of denatured human adenovirus type 18 (Ad 18) DNA can yield up to 80% single-stranded circular molecules which contain a visible projection or "panhandle." In the present report we show that these projections are duplex structures and that they are produced by annealing of the inverted terminal repetition with corresponding complementary sequences at the opposite end of the strand. They thus represent the hydrogen-bonded segments closing circular molecules and consequently provide direct, visual evidence for the arrangement of sequences within the inverted terminal repetition of an adenovirus DNA. MATERIALS AND METHODSViruses and Viral DNA. Human adenovirus serotypes 18 and 31 were prototype strains and were obtained from Flow Laboratories (Rockville, Md.) and H. Shimojo, respectively. Both viruses had been plaque-purified and were free of AAV contamination. Stock pools were prepared by passage in primary human embryonic kidney cells (HEM Research, Inc., Rockville, Md.). Viruses were produced in suspension cultures of KB cells and purified as described (7). The extraction of intact adenovirus DNA from bands of CsCl-purified virus was carried out as before (8)

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A Nuclease from Neurospora Crassa Conidia Specific for Single-Stranded Nucleic Acids

Cited by 40 publications

References 25 publications

Deoxyribonucleic acid strandedness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum.

Deoxyribonucleic acid strandedness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum.

Induction of antibodies to nuclear antigens in rabbits by immunization with hydralazine-human serum albumin conjugates.

Arrangement of sequences in the inverted terminal repetition of adenovirus 18 DNA.

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