The nucleotide sequence of the replication origin of plasmid NTP1.

The sequence of the DNA of the origin region of NTP1 has been obtained. Analysis of the sequence indicates that: (1) there is great sequence homology in the DNA upstream from the origin in NTP1, ColE1, CLODF13, PBR345 AND PBR322; (2) only seven base pairs of NTP1 are identical with the sequence downstream from the origin in ColE1, although some homology exists for 140 bases downstream; (3) two ten base pair direct repeats are present in NTP1 which are also conserved in all four plasmids named above; (4) probably no polypeptide greater than fifteen amino acids in length is encoded by the NTP1 origin region, since no single open reading frame is conserved in all five plasmids.

Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids.

By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm). Plasmid NTP1, which mediates Ap resistance, contains Tn1701. Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm). The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.

Evolution of bacteriophage phi X174. IV. Restriction enzyme cleavage map of St-1.

The St-1 genome is about 6,050 base pairs in size, approximately 10% larger than phiX174 (5,375 base pairs). The DNA fragments obtained by HincII, HaeIII, and EcoRI digestion were ordered and aligned into a colinear map, and the single BglI cleavage site was located.

Evolution of bacteriophage phi C174. V. Alignment of the phi X174, G4, and St-1 restriction enzyme cleavage maps.

The restriciton enzyme cleavage maps of bacteriophage phiS174, G4, and St-1 were aligned by two-dimensional filter hybridization. These studies show that the basic genome structure of phiX174 is conserved in the other two bacteriophage. However, the data also suggest the existence of regions of nonhomology.

Synthesis of R-plasmid-coded beta-lactamase in minicells and in an in vitro system.

beta-Lactamase encoded by a small, nontransferring R-plasmid, NTP1, conferring ampicillin resistance to its host bacteria, was purified. NTP1 plasmid-coded beta-lactamase was found to be periplasmically located in the host Escherichia coli cell, to have a molecular weight of about 25,000, and to show a relatively low activity against oxacillin and methicillin compared with benzylpenicillin. These characteristics indicate that NTP1 plasmid-coded beta-lactamase is very similar or identical to the "TEM-type" beta-lactamase, which is the most common beta-lactamase coded by R-plasmids in enteric bacteria. In minicells containing NTP1 plasmids, at least six plasmid-specific proteins were synthesized, and beta-lactamase was synthesized in a greater amount than other plasmid-coded proteins. In a cell-free transcription-translation coupled system from E. coli, NTP1 plasmid deoxyribonucleic acid directed the synthesis of several species of plasmid-specific proteins, including active beta-lactamase. The in vitro system also showed preferential synthesis of beta-lactamase, as was observed in minicells containing NTP1 plasmids.