Avtor/Urednik		Liang, NS
Naslov		Cloning and expression of toxic phospholipase A2 from Vipera ammodytes ammodytes
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		1993
Obseg		str. 137
Jezik		eng
Abstrakt		A cDNA clone encoding a nontoxic phospholipase A2 (PLAZ), amodytin I2 (amt I2) was isolated from the V a. ammodytes venom gland cDNA library and the nucleotide sequence of 616 bp long insert was determined. The deduced amino acid sequence is 137 residues long, including a 16 amino acid long signal peptide. The codon usage of amt 12 cDNA and PLA2 cDNAs was investigated by comparing cDNAs encoding these proteins. It was found that codon base composition of snake type II; mammalian type II PLA2 and type I PLA2 evolved under strong G/C pressure. The base substitution rate in the first and in the second position of the triplets are higher than in most other cDNAs. This observation suggests that PLA2 isozyme and homologues from V. a. ammodytes evolved without too many evolutionary restriction. Comparison of the cDNA sequences of the PLA2 homologues amt 12, amodyan L (amt L), amodytoxins (amtx) A, B and C reveals that the untranslated region of the cDNAs share a very high degree of similarity, which is higher than the coding region. To the 5'-untranslated region of amt I2 cDNA, a segment with predicted secondary structure in mRNA and a Aff rich region in 3'-untranslated segment were found. This suggests that both untranslated regions of amt 12 mRNA may play some functional role in translation, stability and regulation ofthe mRNAs. A cDNA encoding presynaptically most toxic PLA2 amtxc A from V. a. ammodytes venom has been expressed in E. coli. The amtx A consists of 122 amino acid residues and contains 14 cysteines which form 7 disulphide bonds. Several vector/host systems and forms of expression were tested. The most successful case was in pM AXA/JM109 system where amtx A was expressed as a fusion protein. The fusion gene was composed of a sequence encoding amino acid residues 1-82 of porcine adenylate kinase, followed by DNA sequence encoding 4 amino acid residues long factor Xa recognition and cleavage site and 122 residues of amtx A.(trunc.)
Deskriptorji		VIPER VENOMS PHOSPHOLIPASES A ESCHERICHIA COLI CLONING, MOLECULAR BASE SEQUENCE CODON RECOMBINANT PROTEINS GENE EXPRESSION DNA PLASMIDS DNA, SINGLE-STRANDED NUCLEIC ACID HYBRIDIZATION