Cleavage of Mouse DNA by a Restriction Enzyme as a Clue to the Arrangement of Genes

Botchan, Michael R.; McKenna, Gillies; Sharp, Phillip A.

doi:10.1101/sqb.1974.038.01.041

Cited by 56 publications

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“…2 were compared with the distributions expected on the basis of the theory of Kuhn [18] (Fig.3). The theoretical distributions were drawn by use of the equation presented by Botchan et al [2] and based on calculated 1/P values ( P = probability of doublestrand cleavage by a given enzyme) corrected for a dG + dC content of 36 ' %, in V. ,faha. It is evident from 3.5 pg DNA in restriction buffer containing 5-15 units of the enzymes were incubated in a volume of 10-22 p1 for 6 h at 37 C.…”

Section: Resultsmentioning

confidence: 99%

“…Satellite DNA of these organisms was found to be highly resistant to cleavage by restriction enzymes [2,19] and has been separated after digestion of total DNA from the -bulk of restriction fragments [1,2]. The findings reported in the present paper are similar to those obtained in D. melunoguster by Hamer and Thomas [l] with respect to the occurrence of a highmolecular-weight class of restriction fragments (which these authors called 'spared segments') and their composition of sequences differing in buoyant density.…”

Section: On the Nature O J The Enzyme-resistant Fragmentsmentioning

confidence: 99%

“…In Drosophila and mouse, besides the randomly cleaved bulk DNA, a class of restriction fragments was found to be highly resistant to cleavage. These fragments were shown to be composed of repetitive sequences and proved to be identical to satellite DNA [1,2]. Chromosomal DNA of higher plants has not been the subject of restriction analysis.…”

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confidence: 99%

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Analysis of the Vicia faba Genome by Use of Restriction Endonucleases

Kaina¹,

Hinz²

1979

European Journal of Biochemistry

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DNA of the broad bean, Viciujubu, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BumI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30000 base pairs in length.Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species ( h~ 75 "/).In all digests, a class of high-molecular-weight restriction fragments of more than 30000 base pairs in length was observed which comprised 5 -8 of the genome. It showed hybridization with highly repetitive DNA (c',,t I 2 x lop2 M . s) and included a fraction (2-3'%; of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content : 61 ' %;) containing cleavage sites for the other enzymes used.Among higher organisms, the DNA of Drosophilu melanogaster [l], mouse [2], calf [3,4], and man [5] has been investigated by detailed restriction analysis. In Drosophila and mouse, besides the randomly cleaved bulk DNA, a class of restriction fragments was found to be highly resistant to cleavage. These fragments were shown to be composed of repetitive sequences and proved to be identical to satellite DNA [1,2]. Chromosomal DNA of higher plants has not been the subject of restriction analysis.In this paper we wish to report on an unusual distribution of restriction sites in bulk DNA of Viciu ,faha which significantly deviates from theoretical expectation. Furthermore, we characterized a fragment class which, with respect to its unusually large size, resembles that found in Drosophilu buoyant density similar to that of the total DNA. This observation is of particular interest since in V.Juhu no satellite DNA has been observed after isopycnic sedimentation in CsCl solutions. The findings reported here lead to the isolation of satellite-like DNA of this species which might assist in the study of its organization and evolution in some detail. MATERIALS AND METHODS DNA ExtractionDNA was extracted from root tips of a reconstructed karyotype of Vicia ,faha (termed ACB [6]) by a modification of the technique of Bendich and Bolton [7]. About 20 g of roots were homogenized (14000 rev./ min) in 50 ml ethanol in the cold. The homogenate was washed in 0.15 M NaCl/O.O15 M sodium citrate followed by lysis in 0.3 M NaCl/O.l M EDTAjl 2) sodium dodecyl sulfate, pH 8.0 (30 min, 55 "C). Deproteinization occurred by repeated treatment with phenol/chloroform/isoamyl alcohol (50/50/2). RNA was removed by enzym...

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Section: Resultsmentioning

confidence: 99%

Section: On the Nature O J The Enzyme-resistant Fragmentsmentioning

confidence: 99%

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Analysis of the Vicia faba Genome by Use of Restriction Endonucleases

Kaina¹,

Hinz²

1979

European Journal of Biochemistry

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“…In general the choice of restriction endonuclease has been determined by an empirical approach with the most readily available enzymes tried first until an appropriate enzyme is found. Predictions of the probabilities of restriction endonuclease recognition sites can be used to limit the number of candidate enzymes to those most likely to produce DNA fragments in the size range of interest [5][6][7][8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Prediction of oligonucleotide frequencies based upon dinucleotide frequencies obtained from the nearest neighbor analysis

Hong

1990

Nucl Acids Res

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“…scribed previously (2,11). Cellular DNA was cleaved with various restriction enzymes as described previously (11).…”

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confidence: 99%

BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones.

Pater¹,

Pater²,

Mayorca³

et al. 1982

Mol. Cell. Biol.

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We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecularweight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 pLg/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T-and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH-BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.The human papovavirus BK virus (BKV) was first isolated by Gardner et al. (6). This virus is highly oncogenic in rodents (5, 10,15,20) and transforms rodent cells readily in vitro (9,18). An interesting feature of transformation and tumor induction by this virus is the presence of both integrated and free viral DNA sequences in both transformed and tumor cells (4, 9,13,17,20,22). We have reported recently that viral DNA in hamster brain cells transformed by a closely related variant of this virus, GS (12), is present predominantly in a free form (1). To examine the possibility that viral DNA in at least some of the transformed cells is present without integration, we studied the status of viral DNA in 15 subclones of the parental cell line. We report here that viral DNA in the majority of the cell lines is present only in an integrated fashion. Moreover, the results of our studies of the subclones with integrated viral DNA and DNA treated with mitomycin C indicate that free viral DNA sequences in these cells are most probably the excision products of integrated sequences. These results indicate that although BK virus is similar in its genomic organization to simian virus 40 (7), its mechanism of interaction with the host in transformed cells is more similar to that of polyoma virus (23).MATERALS AND METHODS Cell...

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Cleavage of Mouse DNA by a Restriction Enzyme as a Clue to the Arrangement of Genes

Cited by 56 publications

References 0 publications

Analysis of the Vicia faba Genome by Use of Restriction Endonucleases

Analysis of the Vicia faba Genome by Use of Restriction Endonucleases

Prediction of oligonucleotide frequencies based upon dinucleotide frequencies obtained from the nearest neighbor analysis

BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones.

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