A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

Marko, M A; Chipperfield, R.; Birnboim, H.C.

doi:10.1016/0003-2697(82)90497-3

Cited by 242 publications

(114 citation statements)

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“…Plasmids were prepared by the alkaline lysis procedure (38) and purified additionally as described (39) or isolated by using GeneJET Plasmid Miniprep Kit (Fermentas UAB). Standard techniques (36) were used for recombinant plasmid construction.…”

Section: Methodsmentioning

confidence: 99%

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Jurenaite-Urbanaviciene

Serksnaite²,

Kriukienė

et al. 2007

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

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Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.hybrid ͉ AloI ͉ PpiI ͉ TstI R estriction endonucleases (REases) are parts of restrictionmodification (R-M) systems, whose primary biological function is the protection of bacterial cells from incoming foreign DNA molecules (1). There are three main groups of restriction enzymes (types I, II, and III), which differ in enzyme composition, cofactor requirements, and mode of action (2). The beststudied are type II REases, which in general recognize specific DNA targets of 4-8 bp and cleave DNA at or close to these sequences (1, 2). The exquisite accuracy of type II enzymes (3, 4) has made them indispensable tools for DNA manipulations. Although almost 3,700 type II REases with 262 different specificities have been characterized to date (5), there still is a demand for enzymes recognizing new DNA targets.During the past two decades, numerous efforts have been undertaken to engineer type II REases with altered specificities. Both rational protein design and random mutagenesis, followed by various selection procedures, have been tried (6), and several mutant enzymes with some preference for cleavage of altered DNA targets were isolated (7-10). However, projects concerned with orthodox type II REases so far have been largely unsuccessful mainly for two reasons: (i) difficulty of dealing with the observed tight coupling between DNA recognition and cleavage and (ii) absence of an efficient system for selecting enzymes with changed specificities. In this regard, unorthodox type II enzymes, such as the type IIG REase Eco57I (11), have shown more promise. Type IIG enzymes combine the catalytic centers of endonuclease and methyltransferase in one polypeptide chain, and the ability of Eco57I to methylate recognized DNA targets has been applied to isolate mutants having previously undescribed specificity (12).The discovery of AloI-like REases (13-15), classified as type IIB enzymes, opened up new opportunities for the engineering of type II REases with altered specificities. AloI-like REases ar...

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Section: Methodsmentioning

confidence: 99%

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Jurenaite-Urbanaviciene

Serksnaite²,

Kriukienė

et al. 2007

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

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“…S. flexneri chromosomal DNA was isolated by the method of Marmur (Marmur, ᮊ 1997 Blackwell Science Ltd, Molecular Microbiology, 24, 779-791 Taylor et al (1989) 1961). The method of Kado and Liu (Kado and Liu, 1981) was used to obtain the large virulence plasmid from Shigella, and a modification (Marko et al, 1982) of the alkaline lysis procedure was used to obtain Shigella small plasmids. pHS-2 was separated from the other Shigella plasmids by eluting the DNA band from a 0.8% agarose gel.…”

Section: Methodsmentioning

confidence: 99%

Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Hong

Payne

1997

Molecular Microbiology

117

120

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SummaryThis study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa, rfb, and rol, and in a plasmid-encoded O-antigen chain-length regulator, cld pHS-2 . LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O-antigen side chains or produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX, affected the LPS core region and resulted in reduced amounts of O-antigen. Mutants defective in cld pHS-2 or rol had different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Seré ny test, and serum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cld pHS-2 is required for resistance to serum killing and for full inflammation in the Seré ny test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque formation, but not for serum resistance. Thus, O-antigen synthesis and chain-length regulation genes encoded on both the chromosome and the small plasmid pHS-2 play important roles in S. flexneri invasion and virulence.

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“…Large-scale preparation of E. coli plasmid DNA was done essentially as described by Marko et al. (32) except that CsCl-ethidium bromide gradients replaced purification on glass fibers. Minipreps were made according to Birnboim and Doly (7).…”

Section: Methodsmentioning

confidence: 99%

Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans

et al. 1993

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SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage A integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf6M) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.Streptomyces are gram-positive filamentous soil bacteria notable for their complex morphology as well as the production of a variety of antibiotics and other secondary metabolites, many of which have medical or agricultural importance. Numerous extrachromosomal elements have been identified for these organisms, including a novel family of chromosomally integrated self-transmissible plasmidogenic genetic elements (reviewed in reference 35). SLP1, indigenous to the chromosome of Streptomyces coelicolor (6), was the first of this group to be identified. A model in which the integrated SLP1 element (i.e., SLPlr') can excise from the S. coelicolor chromosome during conjugation with a strain lacking SLP1, transfer to the recipient strain as a transiently existing circular molecule, and either integrate into the recipient chromosome at a site identical to that occupied in the donor strain or undergo deletion of sequences that suppress autonomous replication and consequently be maintained as an autonomously replicating plasmid (33) has been proposed.The recombination reactions mediated by SLP1 are sitespecific and involve 112-bp regions of homology on SLP1 (attP) and the host chromosome (attB) that have only a single-base-pair difference (34). The integration event generates sites at the left (attL) and right (attR) of the integrated element that retain the homologies between attP and attB. Characterization of these sequences (29) integrants. A derivative of SLP1 containing an insertion at a KpnI site 1.3 kb from attP ( Fig. 1), within a locus designated intB, fails to integrate. In addition, integration of SLP1-derived plasmids deleted for a second locus (i.e., intA, Fig. 1) situated at some distance from attP and intB was not observed. This defect could be c...

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A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

Cited by 242 publications

References 7 publications

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans

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