Isolation of a sequence-specific endonuclease (BamI) from Bacillus amyloliquefaciens H

Wilson, Graham K.; Young, Frank E.

doi:10.1016/s0022-2836(75)80028-3

Cited by 302 publications

(53 citation statements)

References 12 publications

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“…After the transformed cells had grown to confluence in the microwells, they were trypsinized, replated at high dilutions, and allowed to grow into macroscopically visible colonies. A (37) was purchased from Bethesda Research Laboratories, Inc. In both cases the DNA concentration in the reaction mixtures was 500 gig/ml or less.…”

Section: Methodsmentioning

confidence: 99%

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

Ketner

Kelly²

1976

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

187

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The DNAs from five independent simian virus 40 (SV40) transformants of the BALB/c 3T3 mouse cell line were digested with either the HpaII or the BamHI restriction endonuclease and the resulting fragments were fractionated by gel electrophoresis. The DNA fragments were denatured in situ in the gel and transferred to a membrane filter. Fragments containing viral DNA were detected by hybridization with high specific activity 32P-labeled SV40 complementary RNA (cRNA) synthesized in vitro. Each of the lines yielded a small number of fragments containing SV40 DNA and the fragments from each line were different. This observation shows that the structure of the integrated SV40 DNA and/or its location in the host DNA are different in each line.A fundamental feature of virus-transformed cells is the persistence of viral genetic information (1-12). In the cases which have been carefully studied to date, the persistent viral genes appear to be covalently linked to host genes (i.e., "integrated"), and expression of one or more integrated viral genes seems to be required for the maintenance of the transformed phenotype (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Little is known at present about the detailed structure of integrated viral DNA or about the molecular mechanisms involved in integration. This is largely due to the fact that viral DNA represents a very small fraction (about 10-6) of transformed cell DNA.In the past several years a good deal of information has accumulated about the structure and function of the genome of the papovavirus simian virus 40 (SV40). Much of this information has been acquired using site-specific bacterial restriction enzymes to dissect the viral DNA and construct detailed physical maps (28). In this paper we show that similar techniques can be used to study the structure of integrated SV40 DNA. Our approach is analogous to that previously employed by Botchan and McKenna (29) Cells and Cell DNAs. BALB/c 3T3 (A317) cells were obtained from G. Todaro and propagated on Eagle's minimum essential medium (MEM) with 20% fetal calf serum. A number of independent SV40-transformed cell lines were derived from these cells by a method similar to that of Todaro and Green (33). BALB/c 3T3 cells growing in 1.6 cm microwells (Linbro) were infected with SV40 (small plaque, strain 776) at a multiplicity of 0.5 PFU per cell after the cells had reached approximately 90% confluence. Twelve to 24 hr after infection the cells were trypsinized and the contents of each microwell were plated in a 100 mm petri dish. Thereafter, the cells were maintained on Eagle's minimum essential medium supplemented with 10% fetal calf serum (MEM-10) which was changed at weekly intervals. Colonies of transformed cells recognizable by their density and altered morphology were first visible at about 2 weeks after infection. The average number of transformed colonies per dish was about three. No transformed colonies were observed in five control dishes containing mock-infected cells. At 3-4 weeks after inf...

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Section: Methodsmentioning

confidence: 99%

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

Ketner

Kelly²

1976

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

187

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“…Potter. Tumors were maintained in mice as described (4 (12), except that 10% glycerol was present in all buffers during the phosphocellulose step. Five milligrams of high-molecular-weight embryo or MOPC 321 tumor DNA in a buffer consisting of 6 mM TrisHCl, pH 7.4,6 mM MgCl2, and 6 mM 2-mercaptoethanol was incubated with 104 units (1 unit is defined as the amount of the enzyme sufficient for digesting 1 ,ug of phage X DNA in 60 min at 370 under the above conditions) of the purified enzyme at 370 for 4 hr.…”

mentioning

confidence: 99%

Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions.

Hozumi¹,

Tonegawa²

1976

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

640

256

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A high-molecular-weight DNA from Balb/c mouse early embryo or from MOPC 321 plasmacytoma (a Kchain producer) was digested to completion with Bacillus amyloliquefaciens strain H restriction enzyme (BamH I). The resulting DNA fragments were fractionated according to size in preparative agarose gel electrophoresis. DNA fragments carrying gene sequences coding for the variable or constant region of K chains were detected by hybridization with purified, 12SI-labeled, whole MOPC 321 K mRNA and with its 3'-end half. The pattern of hybridization was completely different in the genomes of embryo cells and of the plasmacytoma. The pattern of embryo DNA showed two components, one of which (molecular weight = 6.0 million) hybridized with C-gene sequences and the other (molecular weight = 3.9 million) with V-gene sequences. The pattern of the tumor DNA showed a single component that hybridized with both V-gene and C-gene sequences and that is smaller (molecular weight = 2.4 million) than either of the components in embryo DNA. The results were interpreted to mean that the VK and CK genes, which are some distance away from each other in the embryo cells, are joined to form a contiguous polynucleotide stretch during differentiation of lymph~c~es. Such joining occurs in both of the homologous chromosomes. Relevance of these findings with respect to models for V-C gene joining, activation of a specific Vk.gene, and allelic exclusion in immunoglobulin gene loci is discussed. Both light and heavy chains of immunoglobulin molecules consist of two regions: the variable region (V region) and the constant region (C region) (1, 2). Uniqueness (i.e., one copy per haploid genome) of the genetic material coding for C region ("C gene") has been conjectured from normal Mendelian segregation of allotypic markers (3). Nucleic acid hybridization studies have confirmed this notion (4-10). Hybridization studies have also demonstrated that a group of closely related V regions are somatically generated from a few, conceivably even a single, germline gene(s) (V gene) (4-6). (12), except that 10% glycerol was present in all buffers during the phosphocellulose step. Five milligrams of high-molecular-weight embryo or MOPC 321 tumor DNA in a buffer consisting of 6 mM TrisHCl, pH 7.4,6 mM MgCl2, and 6 mM 2-mercaptoethanol was incubated with 104 units (1 unit is defined as the amount of the enzyme sufficient for digesting 1 ,ug of phage X DNA in 60 min at 370 under the above conditions) of the purified enzyme at 370 for 4 hr. In order to investigate completeness of digestion we followed the following scheme. An aliquot of the reaction mixture containing mouse DNA was removed before incubation and mixed with a small amount (ratio of X DNA to mouse DNA, 1:10) of phage X DNA. This pilot mixture was incubated in parallel with the main reaction mixture under the conditions described above. After incubation the pilot mixture was electrophoresed in 0.9% agarose. DNA was visualized under ultraviolet light after staining with 1 ,g/ml of ethidium bromide ...

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“…Endonuclease BamH1 from Bacillus amyloliquefaciens was prepared as in [7]. Oligodeoxyribonucleotides were synthesised by the phosphotriester method [8].…”

Section: Methodsmentioning

confidence: 99%

Restriction endonuclease BamH1 interaction with a synthetic duplex containing half‐size recognition sequences

Zinoviev¹,

Kolesnikov²,

Beznedelnaya³

et al. 1981

FEBS Letters

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Isolation of a sequence-specific endonuclease (BamI) from Bacillus amyloliquefaciens H

Cited by 302 publications

References 12 publications

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions.

Restriction endonuclease BamH1 interaction with a synthetic duplex containing half‐size recognition sequences

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