Nucleotide sequence analysis of the gyrA genes of 10 spontaneous quinolone-resistant gyrA mutants of Escherichia coli KL16, including four mutants examined previously, disclosed that quinolone resistance was caused by a point mutation within the region between amino acids 67 and 106, especially in the vicinity of amino acid 83, of the GyrA protein.Quinolones are considered to exert antibacterial activity by inhibiting DNA gyrase (EC 5.99.1.3), which catalyzes topological changes of DNA (4, 11). DNA gyrase of Escherichia coli consists of subunits A and B, which are the products of the gyrA and gyrB genes, respectively. Mutations in either gene can cause quinolone resistance (4,(15)(16)(17) were determined by dideoxy-chain termination (9) with phage M13mpl8 and M13mpl9 vectors. Table 1 shows the sites and types of mutations and the levels of resistance to quinolones of 10 quinolone-resistant gyrA mutants of E. coli KL16. Four mutants (N-51, P-18, P-10, and N-89) were analyzed previously (17). All 10 point mutations were considered to be solely responsible for quinolone resistance, because replacement of the 0.6-kilobase Sacl-SmaI fragment containing the mutations by the corresponding fragment from wild-type gyrA gene resulted in complete loss of quinolone resistance (data not shown). Sequencing of the 0.6-kilobase SacI-SmaI fragments of the mutant gyrA genes revealed that these mutations were located within a relatively small region (amino acids 67 through 106) of the A subunit, which we call a quinolone resistance-determining region. There were no other mutations in all of the sequenced fragments. Eight of the 10 mutations were in a limited area (amino acids 81 through 87) of the region; surprisingly, five mutations were situated at the same site of amino acid 83. The levels of resistance to quinolones seemed to be related to the mutation sites, because quinolone MICs were high in the decreasing order of MICs for mutants with mutations at amino acids 83, 87, 81, 84, 67, and 106. This result suggests the importance of an area around amino acid 83 of the gyrase A subunit for determining quinolone resistance.Amino acid changes detected at amino acid 83 were Ser to

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The The increase in methicillin-resistant Staphylococcus aureus is a serious problem because only a few effective agents are clinically available. Some quinolones have been used for the treatment of methicillin-resistant S. aureus infections, but the emergence of quinolone resistance has been reported elsewhere (32). Unlike the mechanism underlying the quinolone resistance of gram-negative bacteria such as Escherichia coli (2,7,9,11,12,15,27,31,(36)(37)(38)(39) and Pseudomonas aeruginosa (4,13,16,29,30,36,40)

Angiotensin type 2 receptor gene null mutant mice display congenital anomalies of the kidney and urinary tract (CAKUT). Various features of mouse CAKUT impressively mimic human CAKUT. Studies of the human type 2 receptor (AGTR2) gene in two independent cohorts found that a significant association exists between CAKUT and a nucleotide transition within the lariat branchpoint motif of intron 1, which perturbs AGTR2 mRNA splicing efficiency. AGTR2, therefore, has a significant ontogenic role for the kidney and urinary tract system. Studies revealed that the establishment of CAKUT is preceded by delayed apoptosis of undifferentiated mesenchymal cells surrounding the urinary tract during key ontogenic events, from the ureteral budding to the expansive growth of the kidney and ureter.

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

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