To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive (ts) mutants. Six of these (tsm9, tsm13, tsm20, tsm22, tsm28 and tsm30) failed to replicate in mice and were avirulent. Five mutants (tsm14, tsm18, tsm19, tsm25 and tsm27) were of similar virulence to the parental wild-type (wt) virus, five (tsm7, tsm15, tsm24, tsm26 and tsm31) were 12-100 fold less virulent, five (tsm8, tsm12, tsm16, tsm23 and tsm29) were 150-1500 fold less virulent and four (tsm10, tsm11, tsm17 and tsm21) were between 2,000 and 85,000 fold less virulent than wt. One mutant (tsm28) did not plaque or replicate at 39 degrees C while 5 other mutants (tsm7, tsm9, tsm23, tsm24 and tsm27) also failed to plaque at 39 degrees C but only failed to replicate or replicated poorly at 40 degrees C. A further two mutants (tsm10 and tsm13) were able to plaque and replicate at 39 degrees C but not 40 degrees C. Six other mutants (tsm14, tsm15, tsm16, tsm21, tsm22 and tsm30) failed to form plaques at 40 degrees C and were severely restricted in their replication at 40 degrees C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.

A novel, highly acid-and thermo-stable amylase was detected from culture medium of thermophilic Bacillus strain HUTBS62 isolated from a hot spring located near the Dead Sea, Jordan. The enzyme was purified by precipitation with 60% ammonium sulfate, gel filtration on Sephadex G-100 and DEAE ion exchange chromatography. The enzyme was purified 22.7-fold with 11.6% yield. Purified enzyme was a monomer with a molecular mass of 54.2 kDa. The optimum pH and temperature for catalytic activity was pH 4.4 and 90°C, respectively. Roughly 50% of amylase activity remained even after heat treatment for 90 min at 100°C; moreover 90% of the activity was retained after heat treatment for 2 h at 60°C. The half-life of the enzyme at 70°C, 80°C and 90°C was estimated to be 5, 4 and 2 h, respectively. The activation energy of denaturation of the purified enzyme was 3.29 kJ mol −1 . The presence of 5 mM metal ion affected amylase activity variably; for example: the presence of cobalt, magnesium, cadmium, and manganese increased amylase activity. On the other hand, iron and sodium decreased residual activity to different extents, while calcium, zinc and copper inhibited amylase activity. The enzyme was active in the presence of 1 and 2 mM EDTA at pH 4.4 and 90°C. The purified amylase was acid-and thermo-stable with novel properties making it suitable for many industrial food purposes.

Problem statement:This study reported the purification and characterization of a novel highly thermostable alkaline amylase from a newly isolated Bacillus strain HUTBS71. Approach: The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. Results: Maximum amylase activity (72 U mL −1 ) was obtained at 100°C after 10 min of incubation. The enzyme was purified 24 fold with 12.5% yield and showed a monomer band with a molecular weight of 58.8 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH and temperature, 7.8 and 100°C, respectively. It performed stability over a broad range of pH and temperature, 5.2-10.0 and 80-115°C, respectively. The half-life of the enzyme at 90 and 100°C was estimated to be 3 h. The activation energy of denaturation of purified enzyme was 2.53 kJ moL −1 . The enzyme was activated by 5 mM of CoCl 2 , MgSO 4 , MnCl 2 , ZnSO 4 and MnSO 4 (relative activity was 133, 126, 133, 106.6 and 103%, respectively). It was strongly inhibited by CuSO 4 and CdCl 2 but less affected by NaCl, CaCl 2 , FeCl 3 , ZnCl 2 and EDTA. Conclusion: The present purified amylase therefore could be defined as a highly thermostable, extremely hyperthermophilic and alkalitolerant with new properties make the present enzyme applicable for many starch processing and food industries.

To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive (ts) mutants. Six of these (tsm9, tsm13, tsm20, tsm22, tsm28 and tsm30) failed to replicate in mice and were avirulent. Five mutants (tsm14, tsm18, tsm19, tsm25 and tsm27) were of similar virulence to the parental wild-type (wt) virus, five (tsm7, tsm15, tsm24, tsm26 and tsm31) were 12-100 fold less virulent, five (tsm8, tsm12, tsm16, tsm23 and tsm29) were 150-1500 fold less virulent and four (tsm10, tsm11, tsm17 and tsm21) were between 2,000 and 85,000 fold less virulent than wt. One mutant (tsm28) did not plaque or replicate at 39 degrees C while 5 other mutants (tsm7, tsm9, tsm23, tsm24 and tsm27) also failed to plaque at 39 degrees C but only failed to replicate or replicated poorly at 40 degrees C. A further two mutants (tsm10 and tsm13) were able to plaque and replicate at 39 degrees C but not 40 degrees C. Six other mutants (tsm14, tsm15, tsm16, tsm21, tsm22 and tsm30) failed to form plaques at 40 degrees C and were severely restricted in their replication at 40 degrees C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.

A study of 31 temperature-sensitive mutants of mouse cytomegalovirus has indicated that two mutants (tsm1, tsm31) may be defective in immediate-early/early functions, two (tsm2, tsm3) may be defective in early functions and six (tsm9, tsm18, tsm22, tsm23, tsm28, tsm30) may be defective in early/late functions while the remainder are late function-defective mutants as determined by temperature-shift experiments. Three mutants (tsm1, tsm2, tsm3) were more thermostable than wild-type virus while three (tsm16, tsm26, tsm28) were more thermolabile; the remainder were similar in their thermostability to wild-type virus.