The Ace locus of Drosophila melanogaster has been mapped at the molecular level. cDNA clones from the locus have been isolated and their sequence determined, confirming that Ace forms the structural gene for acetylcholinesterase (AChE). The cDNAs have a 1950 nucleotide open reading frame from which the complete amino acid sequence of AChE has been deduced. The Drosophila enzyme is found to have extensive homology to the known sequence of Torpedo AChE. Ace cDNAs have an unusual structure with a long 5′ leader and several short upstream open reading frames.
Pseudomonas aeruginosa 455, isolated in Ankara, Turkey, produced a pI 6.2 beta-lactamase determined by plasmid pMLH53 and resisted all beta-lactams except carbapenems. This beta-lactamase, named OXA-14, corresponded to OXA-10 (PSE-2) except that aspartate replaced glycine at position 157 and thus is intermediate between OXA-10 and OXA-11, which has aspartate at position 157 and a further substitution at position 143.
Pseudomonas aeruginosa ABD, which was isolated in October 1991 from blood cultures of a burn patient in Turkey, was resistant to cephalosporins, particularly ceftazidime (MIC, 512 pg/ml), penicillins, aztreonam, and meropenem, but not to imipenem. Cephalosporin and penicillin resistance transferred to P. aeruginosa PU21 and was associated with a P-lactamase with a pI of 6.4 encoded by a 100-MDa plasmid designated pMLH52. Like extended-spectrum TEM and SHYV 1-lactamases, this enzyme hydrolyzed penicillins and newer cephalosporins but did not hydrolyze cefoxitin or carbapenems. However, it differed from TEM and SHV derivatives in being a potent oxacillinase, and its encoding gene did not hybridize with probes to TEM and SHV genes. To characterize the enzyme, libraries of total DNA were cloned into plasmid pUC19 and were transformed into Escherichia coli DH5a. Recombinant plasmids that gave ceftazidime resistance all contained a 3.65-kb BamHI fragment. Deletions from this fragment allowed the -lactamase gene to be located on a 1.4-kb section of DNA, which contained an open reading frame of 798 bases. This encoded a protein that was deduced to differ from PSE-2 (3-lactamase only in having serine instead of asparagine at position 143 and aspartate instead of glycine at position 157. It is concluded that the resistance of isolate ABD depended on an extended-spectrum variant of the PSE-2 enzyme. The ability of this enzyme to cause ceftazidime resistance depended primarily on a low K,,, for the compound; Vm,, remained low. It is proposed that PSE-2 should be transferred to the OXA group as OXA-10 and that the new enzyme be designated OXA-11.Gram-negative bacteria have developed several mechanisms for evading the action of expanded-spectrum cephalosporins. Strains ofPseudomonas aeruginosa and Enterobacter, Citrobacter, Serratia, and Morganella spp. resist these compounds by producing elevated amounts of chromosomal class C (Bush group 1) 3-lactamases (17,30). Resistance to expanded-spectrum cephalosporins in other enterobacteria is mostly caused by extended-spectrum variants of the class A ,3-lactamases TEM-1, TEM-2, and SHV-1. These variants differ from their parent enzymes at only one to four amino acid positions but hydrolyze a much wider range of cephalosporins and monobactams (13,26). Since the extendedspectrum TEM and SHV P-lactamases are often encoded by plasmids, they can transfer among species. Currently, they are commonest in Klebsiella pneumoniae, but they have also been reported from Escherichia coli, Proteus mirabilis, Enterobacter spp., Citrobacter freundii, Salmonella spp., and Serratia spp., and their further dissemination seems inevitable (6, 26).Extensive use of newer P-lactams is also selecting other plasmid-mediated extended-spectrum ,-lactamases besides TEM and SHV derivatives. Several extended-spectrum enzymes, including MIR-1, BIL-1, and CMY-2, are plasmidmediated forms of the class C chromosomal
Drosophila melanogaster cells and tissues respond to heat shock by dramatically altering their pattern of transcription and translation, leading to the rapid synthesis of a small number of polypeptides, the heat shock proteins (hsps). By using cloned hsp DNA we have detected sequences complementary to heat shock genes in RNA prepared from non-heat-shocked animals of different developmental stages. Hsp 83 mRNA is present at high levels in all stages examined. Hsp 68 and 70 mRNAs are present at very low levels in most stages and at slightly higher concentration in pupae. Hsp 26 and 27 mRNAs are detected in embryos. Hsp 23, 26 and 27 mRNA are barely detectable in early third instar larvae but are major components of late third instar and early pupal RNA. Hsp 22 mRNA is also detected in early pupae. Later in development the levels of the small hsp mRNAs decrease but a further peak in abundance of hsp 26 and 27 mRNAs is found in mature adult females.
Extended-spectrum beta-lactamases (ESBLs) are an increasing cause of resistance to oxyimino-aminothiazolyl cephalosporins, especially in klebsiellae. In a recent survey we detected ESBLs in 220 (23%) of 966 consecutive klebsiellae from 35 intensive care units (ICUs) in southern and western Europe. The present study examined the extent to which this distribution reflected epidemic strain spread, as against the distribution of ESBL genes into unrelated strains. All 220 ESBL producers were subjected to capsular serotyping and pulsed-field gel DNA electrophoresis (PFGE). Beta-Lactamases were typed for strains isolated on three or more occasions, with the emphasis on SHV enzymes, as these were commoner than TEM variants. Serotyping and PFGE typing defined 85 distinct strains, from 23 of the 35 participating centres. Of 14 centres that contributed five or more ESBL producers, all sent representatives of more than one strain, and two centres sent members of ten or more different strains in contributions of 17-21 ESBL-producing isolates. Nevertheless, epidemic strains-defined as those represented by three or more isolates-accounted for a majority (61%) of the collection. Fifty-two isolates of the same serotype K25 (occasionally acapsular) strain with SHV-4 beta-lactamase were recovered at two French hospitals and one in Belgium. This strain has been found by others in France, and has become particularly widespread. Another single strain was found in two separate Portuguese centres, and many individual hospitals had one or more epidemic strain(s), as well as a scatter of diverse ESBL producers. Major variation in antibiogram and plasmid profile was apparent within strains, with some intra-strain variation in beta-lactamase type. These data imply a fluid situation, with resistance determinants being gained, modified or lost. The endemicity of ESBL producers is disturbing since it limits the potential for control by blocking strain spread, while the diversity within strains is disturbing because it complicates the design of antibiotic policies even during 'single strain' outbreaks.
The rag locus of Porphyromonas gingivalis W50 encodes RagA, a predicted tonB-dependent receptor protein, and RagB, a lipoprotein that constitutes an immunodominant outer membrane antigen. The low G؉C content of the locus, an association with mobility elements, and an apparent restricted distribution in the species suggested that the locus had arisen by horizontal gene transfer. In the present study, we have demonstrated that there are four divergent alleles of the rag locus. The original rag allele found in W50 was renamed rag-1, while three novel alleles, rag-2 to rag-4, were found in isolates lacking rag-1. The three novel alleles encoded variants of RagA with 63 to 71% amino acid identity to RagA1 and each other and variants of RagB with 43 to 56% amino acid identity. The RagA/B proteins have homology to numerous Bacteroides proteins, including SusC/D, implicated in polysaccharide uptake. Monoclonal and polyclonal antibodies raised against RagB1 of P. gingivalis W50 did not cross-react with proteins from isolates carrying different alleles. In a laboratory collection of 168 isolates, 26% carried rag-1, 36% carried rag-2, 25% carried rag-3, and 14% carried rag-4 (including the type strain, ATCC 33277). Restriction profiles of the locus in different isolates demonstrated polymorphism within each allele, some of which is accounted for by the presence or absence of insertion sequence elements. By reference to a previously published study on virulence in a mouse model (M. L. Laine and A. J. van Winkelhoff, Oral Microbiol. Immunol. 13:322-325, 1998), isolates that caused serious disease in mice were significantly more likely to carry rag-1 than other rag alleles.
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