A novel cloning vector for the direct selection of recombinant DNA in E. coli

Hennecke, Hauke; Günther, Isolde; Binder, Florian

doi:10.1016/0378-1119(82)90011-7

Cited by 97 publications

(23 citation statements)

References 13 publications

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“…This enzyme is sensitive to the amino acid analog p-fluorophenylalanine (PFP). E. coli mutant RR28 (17), carrying a mutation in the chromosomal pheS gene, is PFP resistant (PFPr). In the merodiploid situation, the vector-borne, dominant pheS gene needs to be inactivated to allow growth of RR28 cells on medium containing PFP.…”

Section: Methodsmentioning

confidence: 99%

“…1B) (17) and contains the pheS gene subcloned from its original vector, pHWO (11). The AvaI-pheS-BamHI fragment was inserted into the multiple cloning site of pUC8 (43) between Smal and BamHI.…”

Section: Methodsmentioning

confidence: 99%

“…Advantage was taken of two direct selection systems initially constructed to simplify cloning experiments with E. coli (7,17), as well as a system described by Gay and coworkers (15). The common principle of these systems is based upon insertional inactivation of a vectorborne dominant sensitivity marker.…”

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confidence: 99%

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Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

et al. 1991

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On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a dear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliot strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.Insertion sequence (IS) elements are defined as relatively small mobile genetic entities which, unlike drug resistance transposons, do not contain selectable genes (5). This definition makes it clear that direct selection for the presence of an IS element is usually not possible. IS elements in procaryotes were first identified as causative agents of spontaneous mutations in Escherichia coli (21, 36). Since then, various classes of transposable elements have been found to be natural constituents of many bacterial chromosomes, plasmids, and bacteriophages. As far as gram-negative bacteria are concerned, most IS elements have been isolated from enteric bacteria (mainly E. coli; for reviews and lists of isolated elements, see references 14 and 20). However, there have been a substantial number of reports on IS elements identified in other gram-negative bacterial species, such as Agrobacterium tumefaciens (3,15,24,42), Xanthomonas campestris (22, 23), and various Rhizobium (9,28,32) and Pseudomonas (6,13,35,41,45) strains.In general, these elements have been found more or less by chance because of the capacity to inactivate or activate particular genes. However, there have been successful attempts to screen for transposable elements by means of direct selection procedures (8,15,30,35).In this report, we present the construction and use of three broad-host-range vectors designed to enable systematic searches for the presence of transposable elements in gramnegative bacteria. Advantage was taken of two direct selection systems initially constructed to simplify cloning experiments with E. coli (7, 17), as well as a system described by Gay and coworkers (15). The common principle of these systems is based upon insertional inactivation of a vectorborne dominant sensitivity marker. With pSUP104-pheS and pSUP104-rpsL, this leads to drug resistance. Inactivation of pSUP104-sac allows its host to grow on medium containing 5% sucrose. Both of these events are positively selectable phenotypes in the hosts. (A...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

et al. 1991

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“…Rhizobium meliloti 2011 is a wild-type strain and was obtained from A. Kondorosi (Szeged, Hungary). Escherichia coli RR28 was used for transformation in cloning experiments (19). E. coli S17-1 served as the donor for mobilization of pSUP202 derivatives (51).…”

Section: Methodsmentioning

confidence: 99%

Discovery of a rhizobial RNA that is essential for symbiotic root nodule development

1991

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All of the Azorhizobium, Bradyrhizobium, and Rhizobium genes known to be involved in the development of nitrogen-fixing legume root nodules are genes that code for proteins. Here we report the first exception to this rule: the sra gene; it was discovered during the genetic analysis of a Bradyrhizobium japonicum TnS mutant (strain 259) which had a severe deficiency in colonizing soybean nodules. A DNA region as small as 0.56 kb cloned from the parental wild type restored a wild-type phenotype in strain 259 by genetic complementation. The sra gene was located on this fragment, sequenced, and shown to be transcribed into a 213-nucleotide RNA. Results obtained with critical point mutations in the sra gene proved that the transcript was not translated into protein; rather, it appeared to function as an RNA molecule with a certain stem-and-loop secondary structure. We also detected an sra homolog in Rhizobium meliloti which, when cloned and transferred to B. japonicum mutant 259, fully restored symbiotic effectiveness in that strain. We propose several alternative functions for the sra gene product, of which that as a regulatory RNA for gene expression may be the most probable one.

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“…Restriction fragments of genomic DNA were separated by agarose gel electrophoresis. The fragments of those fractions which hybridized to the probe were cloned in the vector pUN121 [lS] and E. coli RR28 [16]. Transformants were screened by Southern hybridization of prepared plasmid DN As.…”

Section: Cnbr Cleuvugrmentioning

confidence: 99%

Sequence of subunit c of the sodium ion translocating adenosine triphosphate synthase of Propionigenium modestum

Ludwig

Kaim

Laubinger

et al. 1990

European Journal of Biochemistry

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The 30 N‐terminal amino acid residues of the purified ATPase c subunit of Propionigenium modestum have been determined. An oligonucleotide mixture was derived from this sequence and used as probe for cloning the corresponding gene in Escherichia coli. The nucleotide sequence of the gene has been determined and compared with those of ATPase c subunits from other bacteria and chloroplasts. Peculiar sequence similarities are found only at the C‐terminus between the c subunits of the ATPases from P. modestum and from Vibrio alginolyticus, another putative Na+‐translocating ATPase.

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A novel cloning vector for the direct selection of recombinant DNA in E. coli

Cited by 97 publications

References 13 publications

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

Discovery of a rhizobial RNA that is essential for symbiotic root nodule development

Sequence of subunit c of the sodium ion translocating adenosine triphosphate synthase of Propionigenium modestum

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