The sequence of the cloned Bacillus amyloliquefaciens subtilisin gene suggested that this secreted serine protease is produced as a larger precursor, designated preprosubtilisin [Wells,

Chronic hepatitis B virus (HBV) infection, which occurs in 1 to 5% of adult infections and up to 30% of pediatric infections, is characterized by high levels of viral replication averaging a daily production of about 10 11 viral particles, hepatic inflammation, necrosis, and ultimately liver failure (36). An estimated 350 million individuals are classified as chronically HBV infected, with the highest concentrations of infection occurring in large parts of Asia and Africa (23).Chronic HBV can be treated with nucleoside analogues that inhibit polymerase activity. Lamivudine was the first licensed polymerase inhibitor, and it results in significant suppression of HBV DNA levels. However, this response, similar to the loss of hepatitis B virus e antigen, is often not sustained upon discontinuation of treatment (11,28). The emergence of viral resistance in 15 to 20% of treated patients per year clearly pinpoints the limitations of this treatment.Newer drugs such as adefovir dipivoxil, entecavir, and telbivudine can result in less resistance, increased suppression of DNA levels, or in somewhat higher levels of hepatitis B virus e antigen loss. Real long-term treatment data with these drugs are, however, limited, and it is unclear how well these responses would be sustained if therapy were withdrawn.

No abstract

Cellulases belong to the large family of glycosyl hydrolases (GHs) and are produced by a variety of bacteria and fungi. These extracellular enzymes act as endoglucanases (EGs), cellobiohydrolases or beta-glucosidases. In this paper, we describe molecular screening for EGs from the GH family 12. Using three homologous sequence boxes deduced from five previously known members of the family, we analysed 22 cellulase-producing fungal strains obtained from a diverse area of the fungal kingdom. Polymerase chain reactions using degenerate primers designed to the homologous protein boxes were used to identify the family 12 homologues. Several fungi showed the presence of multiple versions of the gene, while amino acid sequence analysis showed diversity in 15 novel members of the family, ranging from 26% to 96% similarity. Our sequence analysis shows that the phylogenetic tree of family 12 EGs can be divided into four subfamilies: 12-1 (fungal group I), 12-2 (fungal group II), 12-3 ( Streptomyces group in which Rhodothermus marinus fits) and 12-4 ( Thermophiles group). Erwinia carotovora may form a new subgroup.

The Lactobacillus bulgaricus Il-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'-and 5'-flanking sequences was determined. The amino acid sequence of the 6-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) 0-galactosidase. The cloned P-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus , -galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these PI-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ ,I-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two ,I-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these PI-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic 0-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.Little is known about the specific amino acids involved in the substrate binding and catalysis of the Escherichia coli lacZ P-galactosidase, even though this enzyme has been studied for many years (35). From iodination (8, 15), fluorotyrosine substitution (27), and active-site-directed inhibitor (11,12,23) experiments, Tyr-503 is thought to be the proton-donating species needed for catalysis. Primarily by the use of active-site-directed reagents (11,13)