A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillinresistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to ,(-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria. * Corresponding author. species were identified by Staphyogram (Terumo, Tokyo, Japan), an identification kit, and the coagulase test. S. aureus SR3626, SR3633, SR3636, SR3639, SR3665, SR3681, and SR3716 were used as mecA-positive control organisms (12), and S. aureus ATCC 25923, 209P JC-1, and Smith were used as mecA-negative control organisms.

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A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase-2, was purified from the culture filtrate by S-Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDSPAGE. When synthetic peptides were used as substrates, SFase-2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenylalaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase.The matured protein sequence of SFase-2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. For insight into the three-dimensional structure of SFase-2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P2,2,2, with cell dimensions a = 6.92 nm, b = 7.28 nm, c = 2.99 nm; one molecule was present in the asymmetric unit.Many studies have been done with the serine proteinases produced by Streptomycetes. Streptomyces griseus, in particular, produces many proteinases [l, 21, three of which have been well studied: proteinase A (SGPA) [3], proteinase B (SGPB) [4], and trypsin-like proteinase [5]. These enzymes have been isolated from the commercially available extracellular filtrate, termed Pronase (Kaken Seiyaku, Tokyo) ; their primary structures, biochemical properties and X-ray structures have been reported [6-81. Recently, an acidic-aminoacid-specific proteinase from S. griseus has been described [9, 101. The serine proteinases produced by S. fradiae have been described by Morihara et al. 1111 and Nickerson et al. [12].

We performed molecular cloning of the cDNAs that encode dog and rat pancreatic phospholipases A2 to predict the primary structures of these enzymes. The deduced amino acid sequences exhibited a highly conservative feature which is common to a group of pancreatic phospholipases A2 from various animal species. Furthermore, the structures of the signal sequences of dog and rat pancreatic phospholipases A2 were predicted, although the assignment of the positions cleaved post-translationally is only tentative at the present time.