Integrative suppression — a way to detect homologies between the Escherichia coli chromosome and cloned DNA fragments

Teifel-Greding, J.

doi:10.1016/0378-1097(84)90206-4

Cited by 4 publications

(4 citation statements)

References 5 publications

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“…1, it was possible to show that the frequency of cointegrate formation correlated with the size of the DNA homology present (Table 1, group A). These results support the previous observations of Liljestrom et al (21) and Teifel-Greding (34). When homologous sequences were interrupted by nonhomologous sequences that approached the size of the total homology, the integration frequency was most closely correlated to the average size of the flanking homologies (Table 1, group B).…”

Section: Resultssupporting

confidence: 91%

New method for generating deletions and gene replacements in Escherichia coli

et al. 1989

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We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44°C. Subsequent growth of these cointegrates at 30°C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.

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Section: Resultssupporting

confidence: 91%

New method for generating deletions and gene replacements in Escherichia coli

et al. 1989

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“…1) and are logarithmically proportional to the extent of homology for inserts of between 0.3 and 1.2 kb. Previous studies of integration by recombination in Bacillus subtilis (9,18) and E. coli (18,27,32) revealed either a linear or an exponential shape for the yieldversus-insert-size curve, depending on the experimental system used, which were all different from that presented here (interplasmid recombination [18], suicide [27,32], and thermosensitive [Ts] plasmids [9]). Interestingly, a previous study of SCO integration in Lactococcus lactis, with a Ts plasmid derivative of pWV01 (pG ϩ host), showed a similar log-linear correspondence, although the experimental system used (Ts plasmid delivery vector) was different (4).…”

Section: Discussioncontrasting

confidence: 88%

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes

Leloup¹,

Ehrlich²,

Zagorec³

et al. 1997

Appl Environ Microbiol

144

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Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK ؊ replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 ؋ 10 3 integrants/g of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional ␤-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.

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“…A log-linear relationship between insert size and the frequency of integration was observed for inserts of between 0.35 and 2.5 kb, a result suggesting a constant average probability of recombination per nucleotide pair. Previous studies of integration by recombination in B. subtilis (24) and E. coli (24,34) did not reveal a similar log-linear correspondence. However, as the experimental systems used (interplasmid recombination [24] and suicide [34] and Ts plasmid delivery vectors) were all different, direct comparisons are not meaningful.…”

Section: Resultsmentioning

confidence: 66%

“…Previous studies of integration by recombination in B. subtilis (24) and E. coli (24,34) did not reveal a similar log-linear correspondence. However, as the experimental systems used (interplasmid recombination [24] and suicide [34] and Ts plasmid delivery vectors) were all different, direct comparisons are not meaningful. In a recent study of sco integration in B. subtilis with a Ts derivative of rc plasmid pE194, integration frequency showed a linear dependence on substrate length (14).…”

Section: Resultsmentioning

confidence: 66%

High-efficiency gene inactivation and replacement system for gram-positive bacteria

et al. 1993

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A system for high-efficiency single-and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG'host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37°C. A nested set of L. lactis chromosomal fragments cloned onto pG'host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37°C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28°C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40%o of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.Numerous gram-positive bacteria are targets of study as biological models (e.g., Bacillus subtilis), industrially important fermenter strains (the lactic acid bacteria), or pathogens (e.g., clostridia, listeria, staphylococci, and streptococci). Many strains of industrial or medical importance have been characterized physiologically, but relatively few have been studied or modified genetically. The study or modification of strains could be facilitated by the use of delivery vectors for the introduction of directed or nonspecific insertions in the bacterial chromosome. Delivery systems that rely on nonreplicative vectors are limited to bacteria that can be transformed at a high frequency, and those with conditionally active replicons are often restricted in their host range. Thus, the construction of recombinant strains requires substantial effort and can be applied efficiently only to particular organisms.We previously described a broad-host-range thermosensitive (Ts) plasmid, pVE6002 (22), which was isolated from pGK12 (15). Such plasmids replicate by a rolling-circle (rc) mechanism (rc plasmids) (21). pVE6002 was shown to have potential use as a delivery vector in numerous gram-positive bacteria (22). Ts plasmids are nonreplicative at 37°C, making them particularly useful in bacteria with a low-temperature growth range or when a drastic thermal shock is un...

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Integrative suppression — a way to detect homologies between the Escherichia coli chromosome and cloned DNA fragments

Cited by 4 publications

References 5 publications

New method for generating deletions and gene replacements in Escherichia coli

New method for generating deletions and gene replacements in Escherichia coli

Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes

High-efficiency gene inactivation and replacement system for gram-positive bacteria

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