Interest in whole-genome research has grown substantially over the past few months. This article explores the challenging ethics issues associated with this work.

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Three IS5 elements were mapped in overlapping chromosomal segments on a series of F-prime plasmids by restriction analysis and hybridization. ISSA was located clockwise ofproA near 6 min, ISSB was located clockwise ofpurE near 12 min, and ISSC was tentatively located near 14 min on the Escherichia coli K-12 map. The physical structures of nine type II F-prime plasmids that contain chromosomal DNA from this region indicated that these plasmids were excised from the chromosome by recombination between pairs of IS5 elements.

Insertion element IS121 was mapped between proA and a previously mapped ISSA element in two F-prime plasmids. Results of hybridizations of IS121 to chromosomal DNA from four other strains suggest that IS121 is normally present at this position in the chromosomes of Escherichia coli K-12 strains.Insertion elements are responsible for several types of genome rearrangements that alter the sequence organization of the proA-purE region (-300 kilobase pairs [kb]) of the Escherichia coli K-12 chromosome. IS2 or IS3 elements mediate integration of F in this region (4), IS5 elements mediate type II F-prime excision (19), IS] elements are responsible for specialized transduction of argF by P1 (8,(20)(21)(22), and IS3 elements generate chromosomal inversions (2, 16). These events are reasonably described by recA-dependent recombination between pairs of insertion elements.There are features of the proA-purE region of the chromosome that are not explained by the mechanisms associated with the mapped copies of IS], IS2, IS3, and IS5 in the region. These features include the discontinuities in homologies between the proA-proC regions of E. coli K-12 and Salmonella typhimurium (3,11,14) and some categories of chromosomal deletions.Recently, a new insertion sequence, IS121 (possibly identical to IS30 [9]), has been reported as a component of the Mu dIl(Ap lac) bacteriophage (12). At least three copies of this 1.2-kb element appear in the chromosomes of several E. coli K-12 strains. Because of our interest in the causes for large chromosomal deletions in this region, we used an IS121 probe to screen F-prime plasmids for chromosomal IS121 copies lying in the chromosomal segment from 4 min (92 kb counterclockwise of proA) to the region of the lip gene near 14 min (-500 kb total) as well as an 11-kb region containing the aroG-gal genes (near 17 min). We determined that one IS121 copy is located in this region near proA (6 min).The AtraF proA+ plasmids pRH112 and pRH114 (derived from Hfr strains OR11 and OR72, respectively [5]), the F lac+ proC+ purE+ plasmid ORF4 (derived from Hfr OR11 [2]), and the F aroG+ gal' plasmid F152-1 (derived from Hfr P3 [13]) were digested with EcoRI, and the fragments were resolved electrophoretically and subjected to Southern hybridization analysis with 32P-labeled pMB0321 or pMB0301 as probe. Plasmid pMB0321 contains a BamHI fragment from pSC101::Mu dIl(Ap lac) and includes a copy of IS121. pMB0301 is similar to pMB0321, except that it contains no IS121 (12), and it served as a vector control in hybridization experiments. Plasmid ORF4, which carries chromosomal DNA from ISSA to lip inclusively (7,19), did not hybridize to pMB0321. Plasmid F152-1, which carries at least 14.2 kb of chromosomal sequence clockwise of a434 (near 12 min) * Corresponding author. and about 11 kb of DNA containing the aroG and gal genes (13,19), also failed to hybridize to pMB0321. However, the 5.8-kb EcoRI fragments and the 8.3-kb BamHI fragments from the AtraFproA' plasmids pRH112 (Fig. 1) and pRH114 (data not shown) hybridize...

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.