Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.

Grant, Seth G. N.; Jessee, Joel; Bloom, Fredric R.; Hanahan, Douglas

doi:10.1073/pnas.87.12.4645

Cited by 1,049 publications

(398 citation statements)

References 29 publications

Supporting

Mentioning

393

Contrasting

Unclassified

Order By: Relevance

“…Bacterial conjugations were performed as described (27). Donor and recipient strains were D1210 (28) and DH5␣ (29), respectively. For integration experiments, strains CC118 pir and S17-1 pir (30) were used as donors in matings with strains UB1637 (31) or DH5␣, respectively.…”

Section: Methodsmentioning

confidence: 99%

Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells

Draper

César

Machón

et al. 2005

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

100

139

View full text Add to dashboard Cite

Conjugative relaxases are the proteins that initiate bacterial conjugation by a site-specific cleavage of the transferred DNA strand. In vitro, they show strand-transferase activity on single-stranded DNA, which suggests they may also be responsible for recircularization of the transferred DNA. In this work, we show that TrwC, the relaxase of plasmid R388, is fully functional in the recipient cell, as shown by complementation of an R388 trwC mutant in the recipient. TrwC transport to the recipient is also observed in the absence of DNA transfer, although it still requires the conjugative coupling protein. In addition to its role in conjugation, TrwC is able to catalyze site-specific recombination between two origin of transfer (oriT) copies. Mutations that abolish TrwC DNA strandtransferase activity also abolish oriT-specific recombination. A plasmid containing two oriT copies resident in the recipient cell undergoes recombination when a TrwC-piloted DNA is conjugatively transferred into it. Finally, we show TrwC-dependent integration of the transferred DNA into a resident oriT copy in the recipient cell. Our results indicate that a conjugative relaxase is active once in the recipient cell, where it performs the nicking and strand-transfer reactions that would be required to recircularize the transferred DNA. This TrwC site-specific integration activity in recipient cells may lead to future biotechnological applications.bacterial conjugation ͉ plasmid R388 ͉ site-specific integration ͉ strand transferase ͉ TrwC B acterial conjugation is a widespread mechanism for horizontal DNA transfer among prokaryotes. Under laboratory conditions, conjugation has also been reported between bacteria and eukaryotic cells (1-3). Any DNA molecule containing a short segment called the origin of transfer (oriT) can be conjugatively transferred to a recipient cell if the rest of the conjugation machinery is provided, either in cis or in trans. The transfer apparatus can be divided into three functional modules (4 -6). (i) A nucleoprotein complex known as relaxosome consists of oriT-binding proteins and their cognate DNA. These proteins include one relaxase plus one or more accessory nicking proteins; the relaxase introduces a site-specific nick at the oriT and remains covalently bound to the 5Ј end of the strand that is to be transferred. (ii) A type IV secretion system (T4SS) is a multiprotein complex spanning the inner and outer membranes through which the substrate is secreted. (iii) The conjugative coupling protein (T4CP) is responsible for connecting the two other functional modules. Current models for DNA transfer through T4SS, in both conjugation and the Agrobacterium tumefaciens T-DNA transfer system (7,8), postulate that a T4CP recruits the relaxosome to the T4SS via protein-protein interactions.For Ͼ20 years, models for conjugative DNA transport have postulated that the relaxase catalyzes the final recircularization step of the transferred DNA because of its strand-transfer activity (8, 9). Nevertheless, it was not until r...

show abstract

Section: Methodsmentioning

confidence: 99%

Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells

Draper

César

Machón

et al. 2005

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

100

139

View full text Add to dashboard Cite

show abstract

“…C. glutamicum strains used in this study were obtained from the American Type Culture Collection. E. coli DH5aMCR (Grant et al, 1990) was used as host for recombinant plasmids. E. coli strains were routinely grown at 37 uC on solid Luria broth complex medium (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

et al. 2006

View full text Add to dashboard Cite

The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

show abstract

“…However, up to the present, comprehensive information about the status of the transgene copies in F4 transgenic fish has been lacking. Since the technique of plasmid rescue was firstly described by Perucho et al [13]), it has been utilized in the study of transgenic mice [14][15][16][17], transgenic tomato [18] and transgenic Drosophila [19], but it has rarely, to our knowledge, been employed previously in the study of transgenic fish. Recently, we briefly reported three integration-site sequences in F4 transgenic fish [20]; however, the detailed integration pattern of transgene in F4 transgenic fish needs to be clarified.…”

Section: Introductionmentioning

confidence: 99%

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

Sun

Wang

et al. 2005

Cell Res

View full text Add to dashboard Cite

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

show abstract

Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.

Cited by 1,049 publications

References 29 publications

Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells

Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells

The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

Contact Info

Product

Resources

About