Two R factors, one ( R15 ) conferring resistance to streptomycin and sulfonamide (SM r SU r ) and the other ( 222/R3 ) to streptomycin, sulfonamide, and chloramphenicol (SM r SU r CM r ), were transferred to a Proteus mirabilis strain, and deoxyribonucleic acid (DNA) extracted from these strains was subjected to density-gradient centrifugation. R15 -DNA formed a single satellite band at a density of 1.709 g cm −3 . Electron microscopy of samples from this band showed circular molecules of one type, with a contour length of 18 μm (35 × 10 6 daltons). 222/R3 -DNA formed a satellite band with three peaks at densities 1.708, 1.711 and 1.717 g cm −3 . Electron micrographs revealed circular structures from each band with contour lengths, respectively, of 29 (54 × 10 6 daltons), 36 (68 × 10 6 daltons), and 6 μm (12 × 10 6 daltons). “Supertwisted” forms of several molecular species were found. It is suggested that 222/R3 DNA comprises either a single 36-μm molecule or two individual molecules, 29 and 6 μm in length, and that this may reflect the evolutionary development of R factors.
with an average guanine plus cytosine (GC) base ratio of 50 to 56% (6, 10, 18, 19, 26), and they take the form of circular molecules, a proportion of which are covalently closed circular (CCC) structures which are normally supertwisted. Measurement of the contour length of circular molecules in the absence of supertwists has permitted estimates of the molecular weights of several R factors (6, 18, 19). The numbers of R-factor copies per chromosome have been previously estimated to be two to three in Entero-34
Using SI nuclease assays, we located the sites of initiation of transcription of the j3-lactamase gene on Tnl and Tn2. Transcription in Tn2, like that in Tn3, occurred from the P3 promoter, whereas transcription in Tnl was initiated by two stronger and overlapping promoters, Pa and Pb. The nucleotide sequences of Tnl and Tn2 were determined over a 1,195-base-pair segment constituting most of the sequences of the tnpR and bla genes and the intervening region. There were six base-pair differences between Tnl and Tn3. One in the bla regulatory region accounted for the presence of the Pa and Pb promoters, and another in the bla structural gene is consistent with the isoelectric focusing difference found between the Tnl and Tn3 enzymes. In contrast, there were 24 base-pair differences between Tn2 and Tn3, most of them clustered in one segment of the tnpR gene.A number of transposons which determine TEM-type P-lactamases were originally found by heteroduplex studies (17) to be closely related. They were later distinguished as Tnl, Tn2, Tn3, etc., because of their derivation from different plasmids (6). Since that time, a number of restriction site similarities and differences among them have been identified (5, 28), but since nucleotide sequence determinations have been published only for Tn3, comparisons at the nucleotide level have not been made. The plasmid R6K (18) specifies streptomycin and ampicillin resistance, the latter due to a ,-lactamase (bla) gene, which determines the prototype of the TEM group of ,-lactamases (12), later defined as TEM-1 (20), and which is carried on the transposon Tn2660 (9). Tn2660 is identical to Tn3 in a number of genetic and physiological properties (9, 10, 32) and in its nucleotide sequence over approximately 500 base pairs (bp) at each of the terminal regions (16, 32) and over 500 bp within the noncoding and the amino-terminal regions of the ,-lactamase gene (8).We have recently shown (8) that a mutation in Tn2660 which gives rise to an approximate 10-fold increase in ,-lactamase (bla) resistance is due to a single base-pair substitution at nucleotide 3773, 177 bases upstream of the bla initiation codon (Fig. 1) Tn3, Tn2660, and pBR322 (4, 8, 29, 33 (Fig. 1), was then isolated and used as a source for some of the segments for nucleotide sequence determinations and also for cloning in the vector pRO1614 to produce pRC401. pRO1614 was constructed from pBR322 (26) by inserting into the PstI site a 1.85-kilobase segment derived from a cryptic P. aeruginosa plasmid that permits replication in P. aeruginosa or E. coli hosts. The 1.85-kilobase segment carries the 1-lactamase region downstream from the PstI locus, so that in pRO1614 the P-lactamase gene is reconstituted and is functional, having retained the BamHI-PstI segment of pBR322, i.e., nucleotides 3740 to 4495 of Tn3. The derived plasmid pRC401, in which the 924-bp BamHI-PstI segment from Tnl is substituted for the corresponding segment of pBR322, was used to observe ,B-lactamase expression in P. aeruginosa or E. coli hosts.T...
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