Formation of delta tra F' plasmids: specific recombination at oriT.

Delta tra F' plasmids can be isolated from matings between Hfr donors and recA- recipients, with selection for transfer of proximal chromosomal genes. Previous experiments indicate that F DNA from the neighborhood of the transfer origin up to the proximal junction with the chromosomal DNA is present on these plasmids, together with chromosomal segments, some of which belong to distinct size classes. We have sequenced across the novel joints contained in five delta tra FproA+ plasmids and in five delta tra FpurE+ plasmids, and we have compared these with the F sequence near oriT and with a chromosomal site near purE. The previously reported specificity in formation of some of these classes is confirmed at the nucleotide sequence level. The F DNA in nine of these novel joints extended beyond the nicking sites identified by others in lambda oriT+ bacteriophages up to a position between two sequenced oriT- mutations. Small plasmids containing these novel joints are mobilized in trans by pOX38 at frequencies less than 5 X 10(-7) times the mobilization frequencies for similar plasmids that contain oriT. The relations of these findings to the location of the nicking site at oriT are discussed.

Location of the nick at oriT of the F plasmid.

The oriT locus of the Escherichia coli K12 F plasmid contains a site at which one of the DNA strands is cleaved as a prelude to conjugal transmission to recipient bacteria. We have remapped this site biochemically by using oriT-containing plasmids that were purified from bacteria expressing the F transfer (tra) functions. The strand interruption was found on the transferred strand 137 base-pairs clockwise of the center of the BglII site at 66.7 on the F map. This location is consistent with the locations anticipated from studies of delta traF' plasmids, but it differs from previous results by other investigators. The strand interruption produced a 3'-OH, but the nature of the 5' terminus of the strand on the other side of the nick was not determined. Some DNA sequence motifs in the vicinity of the oriT nick site of F resemble the chromosomal site involved in formation of delta traF'purE plasmids.

Recombination between IS5 elements: requirement for homology and recombination functions.

Intermolecular recombination between two IS5 elements was measured, using bacteriophage lambda recombination vectors, and was compared to recombination between two copies of an SV40 segment cloned into the same vectors. Experiments were conducted in the presence and in the absence of RecA and Red functions, and with the recombining inserts in the same or in reversed orientation. Under all conditions, IS5 elements recombined in a manner similar to the SV40 inserts, indicating that IS-encoded functions did not confer measurable additional intermolecular recombination ability to IS5 in E. coli K-12. Bacteriophages containing reversed IS5 inserts, for which the 16 base pair (bp) termini are identical in 15 positions and which display 12 bp of uninterrupted homology, recombined at approximately the same low frequency under Rec+ and Rec- conditions, indicating that these short homologies were not good substrates for the Rec system. Bacteriophages having reversed inserts recombined better under Red+ than under Red- conditions, but the crossovers were located in nonhomologous regions flanking the element termini. This suggests that 12-bp homologies are not good substrates for the Red system.

Crosses between insertion and point mutations in lambda gene cI: stimulation of neighboring recombination by heterology.

Intragenic recombination between lambda cI point mutations and insertions was studied in four-factor crosses. In crosses between two point mutations, there is a linear relationship between recombination frequency and distance. However, in crosses between an insertion and point mutations, there is additional recombination in the regions 200 base pairs to the right and to the left of the insertion. The recombinational stimulation occurred with IS insertions and also with insertions consisting of HindIII fragments of SV40 and with a deletion that removes part of cI. This indicated that the stimulation was a result of heterology per se rather than of information encoded by the insertions. Either Rec or Red functions are sufficient for enhanced recombination near a heterology. The stimulation is attributed to more frequent resolution of recombinational intermediates in the neighborhood of a heterology. "Stalling" of migrating branches or invading strands at a heterology may increase the probability of local DNA cleavage.

Comparison of IS1, IS2 and IS3 copy number in Escherichia coli strains K-12, B and C.

The number of copies of IS2 and IS1 in the chromosomes of five Escherichia coli K-12 strains and E. coli B and E. coli C has been determined by hybridization. Among these strains, IS1 copy numbers range from 4 to 19 and IS2 copy numbers range from 0 to 12. IS2 is present once in the E. coli B chromosome, but it is absent from E. coli C. The copy numbers of IS3 in the same seven E. coli strains range from 4 to 6.

A partial restriction map of the proA-purE region of the Escherichia coli K12 chromosome.

EcoRI restriction mapping data for fragments larger than 0.7 kb and contained in a 350-kb region of the Escherichia coli K-12 chromosome are presented. 75% of these fragments have been located relative to proA, B, argF, lac, proC, purE, and various insertion sequence elements normally present in this region. BglII and BamHI maps for the regions near argF and purE are also provided.

Specificity in formation of type II F' plasmids.

Eight new F' plasmids derived from Hfr strains in which F is integrated at the chromosomal element alpha 3 beta 3 have been isolated and subjected to restriction enzyme, hybridization, and electron microscope heteroduplex analysis. Plasmids carrying extensive amounts of bacterial deoxyribonucleic acid were produced even though they were obtained by selection for transfer of lac, which is closely linked to F in the parental Hfr strains. Seven plasmids were type II Flac+ proC+ purE+ plasmids, and one was a type I Flac+ proC+ plasmid. Five of the Flac+ proC+ purE+ plasmids contain approximately 284 kilobases of bacterial deoxyribonucleic acid, which is identical for all five within the resolution of the restriction enzyme analysis. Theses results indicate that type II F' plasmids are the predominant tra+ F' type from this region of the Escherichia coli K-12 chromosome and that the recombination events leading to formation of these plasmids exhibit site specificity.

Mapping of IS1 elements flanking the argF gene region on the Escherichia coli K-12 chromosome.

Two directly-repeated IS1 elements have been mapped on the Escherichia coli K-12 chromosome at positions 23.2 kb and 34.5 kb counterclockwise of the IS3 element alpha3beta3 by using F-prime plasmids (including the F lac- proAB+ plasmid F128) that carry different portions of the bacterial chromosome in the purE to proA region. Mapping was accomplished in part by construction of EcoRI, BamHI, and BglII restriction enzyme cleavage maps. Electron microscope heteroduplex and hybridization studies indicate that the chromosomal region flanked by these IS1 elements is completely homologous to the IS1-argF-IS1 region (Tn2901) on the P1argF5 transducing phage (York and Stodolsky, 1981), which suggests that the argF gene region in the usual E. coli K-12 strains has a transposon-like structure.

Sedimentation equilibrium of polydisperse nonideal solutes: a comparison of three approximations.

In the evaluation of sedimentation equilibrium data for polydisperse nonideal solutions, extrapolation procedures are required for the determination of the true solute weight-average molecular weight and of the light-scattering second virial coefficient. In an attempt to decide just which concentration-dependent parameter should be used in making these extrapolations, Van Holde and Williams, J. Polym. Sci., 11, 243 (1953), and Fujita, J. Phys. Chem., 63, 1326 (1959), and ibid., 73, 1759(1969), have derived relations between apparent and true weight-average molecular weights which, starting from approximate forms of the same differential equations, appear to give different working expressions. The present analysis of these results will demonstrate new and perhaps unexpected relations between them.Further, these approximations are discussed in terms of certain of the available experimental data, the purpose being to clarify the conditions under which the behavior of a polydisperse nonideal solution in the ultracentrifuge may be treated as if a nonideal two-component system were involved.

Specificity in the formation of delta tra F-prime plasmids.

Twenty-three independent delta tra F-prime plasmids from three different Escherichia coli K-12 sublines were isolated from Hfr strains whose points of origin coincided with the IS3 element alpha 3 beta 3 or alpha 4 beta 4 in the lac-purE region of the E. coli chromosome. Electrophoretic analysis of plasmid deoxyribonucleic acid digested with EcoRI and hybridization analysis of plasmid deoxyribonucleic acid digested with BglII revealed that at least 14 of these plasmids were formed by processes involving specific bacterial and F loci. Two of the specific bacterial loci involved in delta tra F-prime formation were located at approximately 3.3 and 11.7 min on the E. coli chromosomal map. Two of the delta tra F-prime plasmids contained bacterial deoxyribonucleic acid with circularization endpoints that mapped very near the termini of the IS2 element that is normally located between lac and proC.

Excision of F plasmid sequences by recombination at directly repeated insertion sequence 2 elements: involvement of recA.

The DNA of the F plasmid is joined to bacterial DNA sequences in the F' ORF203 by directly repeated insertion sequence 2 (IS2) elements. The rate of excision of the F plasmid form this F' (presumably by recombination at the directly repeated IS2s) has been estimated in both recA+ and recA- strains. Normal F is produced in the recA+ strain, but is not detected in recA-. The autonomous plasmids produced in the recA- background were F's having deletions. F excision in this particular recA+ case is specific in the sense that the directly repeated IS2s appear to be more active in recombination than similarly disposed IS3 direct repetitions in this F'.

Intrinsic bends and integration host factor binding at F plasmid oriT.

F plasmid oriT DNA extending from the F kilobase coordinate 66.7 (base pair [bp] 1 on the oriT sequence map) rightward to bp 527 was analyzed for intrinsic bends (by permutation assays) and for binding of integration host factor (IHF) (by gel retardation and DNase footprinting). Intrinsic bending of the 527-bp fragment (bend center approximately at bp 240) was represented as a composite of at least two components located near bp 170 and near bp 260. IHF bound primarily to a site extending from bp 165 to 195 and with lower affinity to a site extending from bp 287 to 319. The intrinsic curvature and sequences to which IHF binds (IHF is known to bend DNA) may play a structural role in oriT function.

Enumeration and identification of IS3 elements in Escherichia coli strains.

Escherichia coli K-12 strains ordinarily contain five IS3 elements. Three of these correspond to previously mapped IS3 elements (R. C. Deonier, G. R. Oh, and M. Hu, J. Bacteriol. 129:1129--1140, 1977; S. Hu, E. Ohtsubo, and N. Davidson, J. Bacteriol. 122:749--763, 1975), and two additional IS3 elements are identified. The distribution of IS3 elements among deoxyribonucleic acid fragments generated by digestion with EcoRI indicates a basic pattern from which deviation is detected.