The DNA fragment (3.3 kb) containing the erythromycin resistance determinant was cloned from Escherichia coli Tf481A and sequenced. Deletion and complementation analyses indicated that the expression of high-level resistance to erythromycin requires two genes, mphA and mrx, which encode macrolide 2-phosphotransferase I and an unidentified hydrophobic protein, respectively.We previously reported a new resistance phenotype with resistance to high levels of erythromycin (EM) in clinical isolate Tf481A of Escherichia coli (12). This resistance was caused by inactivation of macrolide antibiotics by macrolide 2Ј-phosphotransferase I [MPH(2Ј)I]. MPH(2Ј)I is an inducible intracellular enzyme and it inactivates macrolides with a 14-membered ring more strongly than those with a 16-membered ring (13).In this report, we describe the cloning and nucleotide sequencing of the Em r determinant. Furthermore, two genes that are required for the expression of high-level resistance to EM are identified.No transfer of the Em r determinant of E. coli Tf481A to another strain was observed by conjugation (13). By using E. coli Tf481A into which the conjugative plasmid RP1 was introduced (4), the Em r determinant was transferred to other E. coli strains by the broth mating method. Restriction analysis of plasmid RP1-EM481 in a transconjugant showed that a 21.9-kb DNA fragment had been inserted into the PstI-D fragment of RP1 (3).From RP1-EM481, we cloned the Em r determinant and obtained pTZ3509 and pTZ3519 by inserting the 4.1-kb PstI fragment and the 3.3-kb BamHI-PstI fragment into the cloning sites of pUC119 (19), respectively, (the fragments were in opposite orientations). Both plasmids conferred high-level resistance to EM and the production of MPH(2Ј)I. By digestion of pTZ3519 and pTZ3509 with exonuclease III (5), deletion derivatives of various sizes were constructed. The nucleotide sequence of the 3.3-kb BamHI-PstI fragment was determined by the dideoxy-chain termination method (15) with Bca BEST DNA polymerase (17). The full sequence of 3,267 bp is presented in Fig. 1 (Fig. 2).To determine the locations of the genes that conferred highlevel resistance to EM, we investigated the activity of MPH (2Ј)I (13) and the MIC of EM (7) for E. coli strains carrying the various deletion derivatives (Fig. 2). These results indicated that the mphA gene for MPH(2Ј)I was located in the region from nucleotide 1630 (breakpoint of ⌬436) to 2884 (breakpoint of ⌬51). ORF2 and ORF3 were located in this region in opposite orientations. The MIC (200 g/ml) for the strain carrying ⌬115 with a deletion from the 5Ј end to nucleotide 510 was much lower than the original MIC of Ͼ3,200 g/ml, despite the fact that ⌬115 contained mphA. The data suggested that some other gene was necessary for the expression of highlevel resistance to EM. A complementation analysis was performed to determine the region that encoded this gene, temporarily designated mrx. The EcoRI-HindIII fragment of ⌬436 containing mphA was subcloned into pBR322 (now called pBR⌬436), whereas th...
In the trophoblast layer of the chorion laeve of human fetal membranes obtained by cesarean section at the month of normal parturition, cells with condensed nuclei could be observed by histochemical examination. Incubating fetal membranes at 37 degrees C in vitro in cultivation medium, the frequency of cells with condensed nuclei increased in the chorion laeve, associating with an increase in DNA fragmentation and the population of in situ TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-X nick end labeling) staining-positive cells. The progressed apoptotic cell death in the chorion laeve in vitro was suppressed by incubation of the tissue in the presence of glucocorticoids, cortisone or hydrocortisone, which was also demonstrated by DNA fragmentation analysis and in situ TUNEL staining. These results reveal that a substantial proportion of trophoblast cells in the chorion laeve of human fetal membranes are induced to undergo apoptosis at the end of pregnancy, and that the apoptosis progresses rapidly in vitro as the incubation period increases. It is suggested that certain hormones such as glucocorticoid, may be related to the regulation of the apoptosis in human fetal membranes.
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