Some myotoxic or neurotoxic PLA 2 s (phospholipases A 2 ) from pit viper venoms contain characteristic N6 substitutions. Our survey of the venoms of more than ten pit viper genera revealed that N6-PLA 2 s exist only in limited Asian pit vipers of two genera, Protobothrops and Gloydius, and exist as either monomers or the basic subunits of heterodimers in some New World pit vipers. For the newly identified N6-PLA 2 s, the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue, whereas the increased serum creatine kinase level assessed their myotoxicities. The purified N6-PLA 2 s from Protobothrops mangshanensis and Gloydius intermedius saxatilis were found to be presynaptic neurotoxins. In contrast, all N6-PLA 2 s from the venoms of Sistrurus miliarius strackeri, S. m. barbouri, Crotalus viridis viridis, C. lepidus lepidus, Cerrophidion godmani and Bothreichis schlegelii were myotoxins without neurotoxicity even in the presence of crotoxin A. Crotoxin-like complexes were for the first time purified from the venoms of Sitrurus catenatus tergeminus, C. mitchelli mitchelli, C. horridus atricaudatus, C. basiliscus and C. durissus cumanensis. The cDNAs encoding six novel N6-PLA 2 s and subunits of the crotoxin-like complex from S. c. tergeminus were cloned and fully sequenced. Phylogeny analysis showed that two structural subtypes of N6-PLA 2 s with either F24 or S24 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius. Calmodulin binds all the N6-PLA 2 s but crotoxin A may inhibit its binding to crotoxin B and to other neurotoxic N6-PLA 2 s. Structure-activity relationships at various regions of the PLA 2 molecules were extensively discussed.

Utilizing Marathon-ready cDNA library and a genespecific primer corresponding to a partial amino acid sequence determined previously, the complete nucleotide sequence for the cDNA of crocalbin, which binds crotoxin (a phospholipase A 2 ) and Ca 2+ , was obtained by polymerase chain reaction. The open reading frame of the cDNA encodes a novel polypeptide of 315 amino acid residues, including a signal sequence of 19 residues. This protein contains six potential Ca 2+ -binding domains, one Nglycosylation site, and a large amount of acidic amino acid residues. The ability to bind Ca 2+ has been ascertained by calcium overlay experiment. Evidenced by sequence similarity in addition, it is concluded that crocalbin is a new member of the reticulocalbin family of calcium-binding proteins.z 1999 Federation of European Biochemical Societies.

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The subtelomere and a portion of the associated telomeric region (together named 3RTAS) of chromosome IIIR from the Arabidopsis thaliana ecotypes Columbia (Col) and Wassilewskija (Ws) were specifically amplified by polymerase chain reaction and subsequently cloned and sequenced. The centromere-proximal portion of 3RTAS from both ecotypes contained two newly identified potential genes, one encoding the chloroplast luminal 19-kDa protein precursor and the other encoding three potential alternatively spliced CCCH-type zinc finger proteins. The telomere-proximal portion of 3RTAS from the Col ecotype contained short duplicated fragments derived from chromosomes I, II, and III, and that from the Ws ecotype contained a duplicated fragment derived from chromosome V. Each duplicated fragment has diverged somewhat in sequence from that of the ectopic template. Small patches of homologous nucleotides were found within the flanking sequences of both the duplicated fragments and the corresponding ectopic template sequences. The structural characteristics of these duplicated fragments suggest that they are filler DNAs captured by non-homologous end joining during double-strand break repair. Our characterization of 3RTAS not only filled up a gap in the chromosome IIIR sequence of A. thaliana but also identified new genes with unknown functions.