Background: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B-and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT.

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55 % homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.To identify enzymes that digest elastin efficiently, we screened for microbial enzymes among alkalophilic bacteria, because local denaturation of elastin under alkaline conditions might facilitate its enzymatic digestion. Alkaline elastase YaB secreted by the alkalophilic Bacillus strain YaB was thus selected and purified (25). The enzyme was characterized as a serine protease of about 25,000 daltons. Chemical modification studies revealed that certain histidine and tyrosine residues are involved in the enzymatic activity (21). The enzyme has the peculiar characteristic of a very high optimum pH for digestion of casein and elastin and an elastolytic activity comparable to that of porcine pancreatic elastase (25). The substrate specificity was examined by cutting oxidized insulin A and B chains (23) and by using synthetic peptide substrates (24). The enzyme has a P1 substrate preference for Ala, in contrast to that of subtilisin BPN' for Tyr, and it efficiently hydrolyzes succinyl-AlaAla-Ala-p-nitroanilide, a specific substrate of porcine elastase (24). Furthermore, the enzyme binds to elastin below its isoelectric point (pl 10.6) (22). These features encouraged us to characterize the enzyme as a microbial elastase.To determine the molecular mechanism of the extremely high optimum pH and high elastolytic activity of the enzyme, we cloned the structural gene and analyzed its nucleotide and deduced amino acid sequences.Bacillus subtilis DB104 (nprEI8 nprR2 AaprE3 his-101) (9) has lesions in the structural genes for neutral and alkaline proteases and was a gift from Roy H. Doi and Fujio Kawamura. Escherichia coli MC1061 and JM109 were used as the host strains for cloning. All bacterial strains were propagated at 37°C in nutrient broth (Eiken Chemical Co., Tokyo, Japan) or Luria broth (11) with vigorous aeration or on media solidified with 1.5% agar. Media were s pplemerted with 100 p.g of ampicillin per ml, 10 ,ug of kana.nycin pei :nl, or 20 .'g of tetracycline per ml when necessary. pL"U18 and pHY300PLK were purchased from Takara Shuzo Co. (Kyoto, Japan).To detect the production of alkaline elastase, elastin plates and skim milk plates were used. Elastin plates were made by overlaying 5 ml of 1% elastin (Sigma Chemical Co., St. Louis, Mo.) and 0.7% agar on basal agar plates containing * Corresponding author. 0.8% nutrient broth and 1.5% agar. Skim milk plates contained 0.8% nutrient broth, 1.5% agar, and 1% skim milk....

Background: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B-and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an antiTdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex.

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